Methods for preventing and treating A-beta oligomer-associated and/or -induced diseases and conditions

ABSTRACT

The disclosure pertains to methods of treating or preventing a disease or condition associated with and/or induced by soluble A-beta oligomer such as Alzheimer&#39;s disease by administering to a subject in need thereof conformation specific and/or selective antibodies or binding fragments thereof and related products.

RELATED APPLICATIONS

This application is a continuation in part of PCT/CA2016/051300, filedNov. 9, 2016; PCT/CA2016/051305, filed Nov. 9, 2016; PCT/CA2016/051303,filed Nov. 9, 2016; and PCT/CA2017/050866, filed Jul. 18, 2017. Thisapplication also claims priority to U.S. provisional application Ser.No. 62/443,766, filed Jan. 8, 2017; 62/507,633, filed May 17, 2017; and62/507,587, filed May 17, 2017. All of these applications areincorporated herein by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing“7685-P53259US02_SequenceListing.txt” (2,328 bytes), submitted viaEFS-WEB and created on Nov. 9, 2017, is herein incorporated byreference.

FIELD

The present disclosure relates to methods for treating or inhibitingamyloid-beta oligomer related diseases and particularly to methodsexploiting the use of conformational A-beta epitopes that were predictedand shown to be selectively accessible in A-beta oligomers.

BACKGROUND

Amyloid-beta (A-beta), which exists as a 36-43 amino acid peptide, is aproduct released from amyloid precursor protein (APP) by the enzymes βand γ secretase. In AD patients, A-beta can be present in solublemonomers, insoluble fibrils and soluble oligomers. In monomer form,A-beta exists as a predominantly unstructured polypeptide chain. Infibril form, A-beta can aggregate into distinct morphologies, oftenreferred to as strains. Several of these structures have been determinedby solid-state NMR.

For, example, structures for several strains of fibrils are available inthe Protein Data Bank (PDB), a crystallographic database of atomicresolution three dimensional structural data, including a 3-foldsymmetric Aβ structure (PDB entry, 2M4J); a two-fold symmetric structureof Aβ-40 monomers (PDB entry 2LMN), and a single-chain, parallelin-register structure of Aβ-42 monomers (PDB entry 2MXU).

The structure of 2M4J is reported in Lu et al [8], and the structure of2MXU is reported in Xiao et al [9]. The structure of 2LMN is reported inPetkova et al [10].

A-beta oligomers have been shown to kill cell lines and neurons inculture and block a critical synaptic activity that subserves memory,referred to as long term potentiation (LTP), in slice cultures andliving animals.

The structure of the oligomer has not been determined to date. Moreover,NMR and other evidence indicates that the oligomer exists not in asingle well-defined structure, but in a conformationally-plastic,malleable structural ensemble with limited regularity. Moreover, theconcentration of toxic oligomer species is far below either that of themonomer or fibril (estimates vary but are on the order of 1000-foldbelow or more), making this target elusive.

Antibodies that bind A-beta have been described.

WO2009048538A2 titled USE OF ANTI-AMYLOID ANTIBODY IN OCULAR DISEASESdiscloses chimeric antibodies that recognize one or more binding siteson A-beta and are useful for the treatment for ocular diseases.

U.S. Pat. No. 9,221,812B2 titled COMPOUNDS FOR THE TREATMENT OF DISEASESASSOCIATED WITH AMYLOID OR AMYLOID-LIKE PROTEINS describespharmaceutical compositions and discontinuous antibodies that bindA-beta including an epitope between amino acid residues 12 to 24 for thetreatment of amyloid-related diseases.

WO2003070760A2 titled ANTI-AMYLOID BETA ANTIBODIES AND THEIR USEdiscloses antibodies that recognize an A-beta discontinuous epitope,wherein the first region comprises the amino acid sequence AEFRHDSGY ora fragment thereof and wherein the second region comprises the aminoacid sequence VHHQKLVFFAEDVG or a fragment thereof.

US20110171243A1 titled COMPOUNDS TREATING AMYLOIDOSES discloses apeptide mimotope capable of inducing the in vivo formation of antibodiesthat bind HQKLVF and/or HQKLVFFAED, and its use.

WO2008088983A1 and WO2001062801A2 disclose a pegylated antibody fragmentthat binds A-beta amino acids 13-28 (HHQKLVFFAEDVGSNK) and its use intreating A-beta related diseases.

WO2009149487A2 titled COMPOUNDS FOR TREATING SYMPTOMS ASSOCIATED WITHPARKINSON'S DISEASE describes compounds comprising a peptide havingbinding capacity for an antibody specific for an A-beta epitope such asEVHHQKL, HQKLVF and HQKLVFFAED.

The HHQK (SEQ ID NO: 1) domain is described as involved in plaqueinduction of neurotoxicity in human microglia, as described in Giulian Det al. [11] and Winkler et al. [12]. Non-antibody therapeutic agentsthat bind HHQK (SEQ ID NO: 1) have been disclosed for the treatment ofprotein folding diseases (US20150105344A1, WO2006125324A1).

WO2010128139A1 titled BIOMARKERS AND METHODS FOR DIAGNOSING ALZHEIMER'SDISEASE AND/OR MILD COGNITIVE IMPAIRMENT discloses a diagnostic methodfor Alzheimer's disease through assessing levels of antibodies capableof binding pGlu A-Beta in a given subject's body fluid.

WO2011033046A1 titled NOVEL ASSAY FOR THE DETECTION OF AMYLOID BETAPEPTIDES discloses a method for detection of A-beta (1-40).

WO2014161875A1 titled METHOD FOR DETECTING Aβ-SPECIFIC ANTIBODIES IN ABIOLOGICAL SAMPLE discloses a method for detecting A-beta-specificantibodies using A-beta variants for the diagnosis of Alzheimer'sdisease.

U.S. Pat. Nos. 5,766,846; 5,837,672; and 5,593,846 (which areincorporated herein by reference) describe the production of murinemonoclonal antibodies to the central domain of the Aβ peptide. WO01/62801 describes antibodies that bind A-beta between amino acids13-28. WO2004071408 discloses humanized antibodies. WO2008088983A1describes an antibody fragment that binds amyloid beta (A-beta) peptideand is covalently attached to one or more molecules of polyethyleneglycol (PEG), the antibody fragment specifically binding human A-betapeptide between amino acid positions 13-28. Solanezumab and Crenezumabbind amino acids 16-26 on A-beta. Crenezumab interacts with the monomer,oligomer and fibril. Midregion antibodies, including solanezumab(picomolar affinity) and crenezumab (nanomolar affinity), appear topreferentially bind monomeric A-beta [16].

Antibodies that preferentially or selectively bind A-beta oligomers andinhibit A-beta oligomerization are desirable for therapeuticintervention.

SUMMARY

Described herein and in International Application WO2017/079833 filedNov. 9, 2016 and International Application PCT/CA2017/050866 filed Jul.18, 2017 each of which are incorporated herein by reference areconformational epitopes in A-beta comprising and/or consisting ofresidues HHQK (SEQ ID NO: 1) or a part thereof, and antibodies thereto.Also described herein and in International application WO2017/079835filed Nov. 9, 2016, incorporated herein by reference, are conformationalepitopes in A-beta comprising and/or consisting of residues QKLV (SEQ IDNO: 5) or a part thereof, and antibodies thereto. Further describedherein and in International application WO2017/079831 filed Nov. 9, 2016incorporated herein by reference, are conformational epitopes in A-betacomprising and/or consisting of residues HDSG (SEQ ID NO: 9) or a partthereof, and antibodies thereto.

Unlike some other A-beta antibodies, antibodies raised to immunogeniccyclic conformations of the epitopes HHQK (SEQ ID NO: 1), QKLV (SEQ IDNO: 5) or HDSG (SEQ ID NO: 9) specifically bound oligomeric A-beta inpreference to monomeric or fibril forms (see for example FIG. 8). Asdemonstrated herein, antibodies that specifically and/or selectivelybind said conformational epitopes inhibit and/or prevent neurotoxicityand memory deficits induced by soluble A-beta oligomers in a mousemodel.

Injection of oligomer forms of A-beta into the brains of mice causes aneurological deficit that can be assessed in a memory-behavior testcalled novel object recognition as described further below. Normal miceexposed to an object remember the familiar object when re-exposed to itand spend more time exploring a newly introduced object. In contrast,A-beta oligomer-injected mice lose the ability to discriminate betweenknown and novel objects and spend equivalent amounts of time exploringboth. Results obtained in this assay showed that administration to miceof an antibody that selectively binds cyclo(CGHHQKG) (SEQ ID NO: 2),cyclo(CGQKLVG) (SEQ ID NO: 6) or cyclo(CGHDSGG) (SEQ ID NO:10) inhibitedthe loss of short-term memory formation caused by injection of solubleA-beta oligomers.

Accordingly, provided herein are methods for treating or preventing adisease or condition associated with and/or induced by soluble A-betaoligomer comprising administering to a subject in need thereof acompound, immunogen, composition, antibody, nucleic acid or celldescribed herein.

In an embodiment, the method of treating or preventing a disease orcondition associated with and/or induced by soluble A-beta oligomercomprises administering to a subject in need thereof: an isolatedconformation specific and/or selective antibody or binding fragmentthereof that specifically and/or selectively binds to a cyclic compoundcomprising an A-beta peptide having a sequence of QKL, HQK, KLV, HHQK(SEQ ID NO: 1), QKLV (SEQ ID NO: 5) or HDSG (SEQ ID NO: 9), they cycliccompound optionally having a sequence of SEQ ID NO: 2, 3, 4, 6, 7, 8,10, 11 or 12; an immunogen comprising a cyclic compound comprising anA-beta peptide having a sequence of QKL, HQK, KLV, HHQK (SEQ ID NO: 1),QKLV (SEQ ID NO: 5) or HDSG (SEQ ID NO: 9); a cell expressing saidantibody or binding fragment thereof; or a nucleic acid encoding saidantibody or binding fragment thereof.

In an embodiment, the disease or condition associated with and/orinduced by soluble A-beta oligomer is cognitive deficits, optionallyloss of short term memory formation.

In an embodiment, the disease or condition associated with and/orinduced by soluble soluble A-beta oligomer A-beta is Alzheimer's disease(AD).

In an embodiment, the treatment is prophylactic treatment. For examplein an embodiment, the antibody is administered to a subject with apredisposition to developing a disease or condition associated withand/or induced by soluble A-beta oligomer or showing an early sign of ADpathology. For example, AD pathology develops first in the perirhinaland enthorinal cortex before the hippocampus.

In an embodiment, the disease or condition is perirhinal cortexdysfunction or pathology. In another embodiment, the disease orcondition is enthorinal cortex dysfunction or pathology.

In an embodiment, the disease or condition is associated with and/orinduced by soluble A-beta 1-42 oligomer.

In an aspect, an immunogen described herein is administered for treatingor preventing a disease or condition associated with and/or induced bysoluble A-beta oligomer.

In an aspect the immunogen comprises a cyclic compound which comprises:an A-beta peptide the peptide comprising HQK and up to 6 A-betacontiguous residues, and a linker, wherein the linker is covalentlycoupled to the A-beta peptide N-terminus residue and the A-betaC-terminus residue.

In another aspect, the cyclic compound comprises: an A-beta peptide, theA-beta peptide comprising QKL and up to 8, 7 or 6 A-beta residues, and alinker, wherein the linker is covalently coupled to the A-beta peptideN-terminus residue and the A-beta C-terminus residue.

In yet another aspect, the cyclic compound comprises: an A-beta peptide,the A-beta peptide comprising HDSG and up to 8, 7 or 6 A-beta residues,and a linker, wherein the linker is covalently coupled to the A-betapeptide N-terminus residue and the A-beta C-terminus residue.

In an embodiment, the A-beta peptide is selected from a peptide having asequence of any one of SEQ ID NOS: 1, 5, 10, 202 or a part thereof.

In another embodiment, the cyclic compound is cyclic peptide.

In another embodiment, the linker comprises or consists of 1-8 aminoacids, optionally, 3, 4 or 5, and/or equivalently functioning moleculesand/or one or more functionalizable moieties.

In another embodiment, the linker amino acids are selected from A and G,and/or wherein the functionalizable moiety is C. In another embodiment,the linker comprises or consists of amino acids GCG or CGC.

In another embodiment, the linker comprises a PEG molecule.

In another embodiment, the cyclic compound is selected from thestructures in FIG. 1A-FIG. 1C.

An aspect includes an immunogen comprising the cyclic compoundoptionally for treating or preventing a disease or condition associatedwith and/or induced by soluble A-beta oligomer. In an embodiment, thecompound is coupled to a carrier protein or immunogenicity enhancingagent.

In another embodiment, the carrier protein is bovine serum albumin (BSA)or the immunogenicity-enhancing agent is keyhole Keyhole LimpetHaemocyanin (KLH).

In another aspect a composition comprising the immunogen describedherein is administered.

In an embodiment, the composition described herein, further comprises anadjuvant.

In another embodiment, the adjuvant is aluminum phosphate or aluminumhydroxide.

In another aspect an isolated conformation specific and/or selectiveantibody that specifically and/or selectively binds to an A-beta peptidehaving a sequence of HQK, HHQK (SEQ ID NO: 1), HQKL (SEQ ID NO: 202),QKL, QKLV (SEQ ID NO: 5), HDSG (SEQ ID NO: 9) or a related epitopesequence presented in a cyclic compound described herein, is provided.In embodiments said antibody is administered in a method describedherein.

In another embodiment, the antibody selectively binds to a cycliccompound comprising HHQK (SEQ ID NO: 1), QKLV (SEQ ID NO: 5), HDSG (SEQID NO: 9) over a corresponding linear peptide, optionally wherein theantibody is at least 2 fold, 3 fold, at least 5 fold, at least 10 fold,at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold,at least 100 fold, at least 500 fold, at least 1000 fold more selectivefor the cyclic compound over the corresponding linear compound.

In another embodiment, the antibody selectively binds A-beta oligomerover A-beta monomer and/or A-beta fibril.

In another embodiment, the selectivity is at least 2 fold, at least 3fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30fold, at least 40 fold, at least 50 fold, at least 100 fold, at least500 fold, at least 1000 fold more selective for A-beta oligomer overA-beta monomer and/or A-beta fibril.

In another embodiment, the antibody does not specifically and/orselectively bind a linear peptide comprising sequence HHQK (SEQ IDNO: 1) or a related epitope, optionally wherein the sequence of thelinear peptide is a linear version of a cyclic compound used to raisethe antibody, optionally a linear peptide having a sequence as set forthin SEQ ID NO: 2.

In another embodiment, the antibody does not specifically and/orselectively bind a linear peptide comprising sequence QKLV (SEQ ID NO:5) or a related epitope, optionally wherein the sequence of the linearpeptide is a linear version of a cyclic compound used to raise theantibody, optionally a linear peptide having a sequence CGQKLVG (SEQ IDNO: 6).

In another embodiment, the antibody does not specifically and/orselectively bind a linear peptide comprising sequence HDSG (SEQ ID NO:9) or a related epitope, optionally wherein the sequence of the linearpeptide is a linear version of a cyclic compound used to raise theantibody, optionally a linear peptide having a sequence SEQ ID NO: 10.

In another embodiment, the antibody lacks or has negligible binding toA-beta monomer and/or A-beta fibril plaques in situ.

In another embodiment, the antibody is a monoclonal antibody or apolyclonal antibody.

In another embodiment, the antibody is a humanized antibody, optionallycomprising a variable domain described in Table 12 or 13.

In another embodiment, the antibody is an antibody binding fragmentselected from Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers,nanobodies, minibodies, diabodies, and multimers thereof.

In an embodiment, the antibody comprises a light chain variable regionand a heavy chain variable region, optionally fused, the heavy chainvariable region comprising complementarity determining regions CDR-H1,CDR-H2 and CDR-H3, the light chain variable region comprisingcomplementarity determining region CDR-L1, CDR-L2 and CDR-L3 and withthe amino acid sequences of said CDRs comprising the sequences in Table9 A to I or Table 15.

For example, in an embodiment, the antibody comprises a light chainvariable region and a heavy chain variable region, optionally fused, theheavy chain variable region comprising complementarity determiningregions CDR-H1, CDR-H2 and CDR-H3, the light chain variable regioncomprising complementarity determining region CDR-L1, CDR-L2 and CDR-L3and with the amino acid sequences of said CDRs comprising the sequences:SEQ ID NOs: 20-25, SEQ ID NOs: 26-31, SEQ ID NOs: 32-37, SEQ ID NOs:32-34 and 38-40; SEQ ID NOs: 41-46; SEQ ID NOs: 47-52, SEQ ID NOs:53-58, SEQ ID NOs: 59-64, SEQ ID NOs: 59-61 and 65-67; SEQ ID NOs: 59-61and 68-70; SEQ ID NOs: 71-76, SEQ ID NOs: 71-73 and 77-79; SEQ ID NOs:71-73 and 80-82 and SEQ ID NOs: 185-190.

In an embodiment, the antibody or binding fragment comprises a heavychain variable region comprising: i) an amino acid sequence of a heavychain variable sequence as set forth in Table 10; ii) an amino acidsequence with at least 50%, at least 60%, at least 70%, at least 80% orat least 90% sequence identity to said heavy chain variable sequence setout in Table 10, 12 or 13, wherein the CDR sequences are thecorresponding CDRs as set forth in Table 9, or iii) a conservativelysubstituted amino acid sequence i) wherein the CDR sequences are thecorresponding CDRs as set forth in Table 9; and a light chain variableregion comprising i) an amino acid sequence of a light chain variablesequence as set forth in Table 10, ii) an amino acid sequence with atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%sequence identity to said light chain variable sequence as set out inTable 10, 12 or 13, wherein the CDR sequences are the corresponding CDRsas set forth in Table 9, or iii) a conservatively substituted amino acidsequence i) wherein the CDR sequences a are the corresponding CDRs asset forth in Table 9.

In an embodiment a combination of antibodies and/or binding fragmentsthereof described herein are administered.

In another embodiment, the antibody of binding fragment competes forbinding to human A-beta with an antibody comprising the CDR sequence set(e.g. CDR H1-3 and CDR L1-3) as recited herein, for example in Table 9,the variable sequence set (e.g. Heavy chain and light chain variableregions, for example in Table 10, 12 or 13) and/or antibody produced bythe hybridoma cell line deposited under the provisions of the BudapestTreaty with the American Type Culture Collection (ATCC®) 10801University Blvd., Manassas, Va., 20110-2209, USA on Jul. 19, 2017 andgiven the Accession number PTA-12431.

In yet another aspect an immunoconjugate comprising the antibody orbinding fragment described herein and a detectable label or cytotoxicagent. In embodiments, said immunoconjugate is administered in methodsprovided herein.

An aspect includes a composition comprising the antibody or bindingfragment described herein, or the immunoconjugate described herein aswell as combinations of two or more antibodies or binding fragmentsthereof described herein, optionally with a diluent. In embodiments, thecomposition is administered in methods provided herein

In yet a further aspect a nucleic acid molecule encoding a proteinaceousportion of the compound or immunogen described herein, the antibodydescribed herein or proteinaceous immunoconjugates described herein. Insome embodiments the nucleic acid molecule is administered in methodsprovided herein.

In another aspect a vector comprising the nucleic acid described hereinis provided and optionally administered in methods provided herein.

In another aspect a cell expressing an antibody or binding fragmentdescribed herein is provided. In embodiments, said cell is administeredin methods provided herein.

Also provided is a hybridoma cell line deposited under the provisions ofthe Budapest Treaty with the American Type Culture Collection (ATCC®)10801 University Blvd., Manassas, Va., 20110-2209, USA on Jul. 19, 2017and given the Accession number PTA-12431.

An aspect includes a kit comprising the compound described herein, theimmunogen described herein, an antibody described herein, theimmunoconjugate described herein, the composition described herein, thenucleic acid molecule described herein, the vector described herein orthe cell described herein optionally for use in treating or preventing adisease or condition associated with and/or induced by soluble A-betaoligomer.

An aspect includes a method of making the antibody described herein,comprising administering the compound or immunogen described herein or acomposition comprising said compound or immunogen to a subject andisolating antibody and/or cells expressing antibody specific orselective for the compound or immunogen administered and/or A-betaoligomers, optionally lacking or having negligible binding to a linearpeptide comprising the A-beta peptide and/or lacking or havingnegligible plaque binding for use in treating or preventing a disease orcondition associated with and/or induced by soluble A-beta oligomer.

An aspect includes a method of determining if a biological samplecomprises A-beta, the method comprising:

-   -   a. contacting the biological sample with an antibody described        herein or the immunoconjugate described herein; and    -   b. detecting the presence of any antibody complex.

In another embodiment, the method comprises:

-   -   a. contacting the sample with the antibody described herein or        the immunoconjugate described herein that is specific and/or        selective for A-beta oligomers under conditions permissive for        forming an antibody: A-beta oligomer complex; and    -   b. detecting the presence of any complex; the presence of        detectable complex is indicative that the sample may contain        A-beta oligomer; and    -   c. optionally treating the subject.

In another embodiment, the amount of complex is measured.

In another embodiment, the sample comprises brain tissue or an extractthereof, whole blood, plasma, serum and/or CSF.

In another embodiment, the sample is a human sample.

In another embodiment, the sample is compared to a control, optionally aprevious sample.

In another embodiment, the level of A-beta is detected by SPR.

An aspect includes a method of measuring a level of A-beta oligomers ina subject, the method comprising administering to a subject at risk orsuspected of having or having AD, an immunoconjugate comprising anantibody described herein wherein the antibody is conjugated to adetectable label; and detecting the label, optionally quantitativelydetecting the label.

In an embodiment, the label is a positron emitting radionuclide.

An aspect includes a method of inducing an immune response for treatingor preventing a disease or condition related with and/or induced bysoluble A-beta oligomer in a subject, comprising administering to thesubject a compound or combination of compounds described herein,optionally a cyclic compound comprising HQK or HHQK (SEQ ID NO: 1), QKL,QKLV (SEQ ID NO: 5) or HDSG (SEQ ID NO: 9) or a related epitope peptidesequence, an immunogen and/or composition comprising said compound orsaid immunogen; and optionally isolating cells and/or antibodies thatspecifically or selectively bind the A-beta peptide in the compound orimmunogen administered.

An aspect includes a method of inhibiting A-beta oligomer propagationfor treating or preventing a disease or condition associated with and/orinduced by soluble A-beta oligomer, the method comprising contacting acell or tissue expressing A-beta with or administering to a subject inneed thereof an effective amount of an A-beta oligomer specific orselective antibody, binding fragment or immunoconjugate describedherein.

Another aspect includes a method of treating AD and/or other A-betaamyloid associated and/or induced diseases and conditions, the methodcomprising administering to a subject in need thereof i) an effectiveamount of an antibody or immunoconjugate described herein, optionally anA-beta oligomer specific or selective antibody, or a pharmaceuticalcomposition comprising said antibody; 2) administering an isolatedcyclic compound comprising HQK, HHQK (SEQ ID NO: 1), QKL, QKLV (SEQ IDNO: 5) or HDSG(SE ID NO: 9) or a related epitope sequence or immunogenor pharmaceutical composition comprising said cyclic compound, or 3) anucleic acid or vector comprising a nucleic acid encoding the antibodyof 1 or the immunogen of 2, to a subject in need thereof.

In an embodiment, a biological sample from the subject to be treated isassessed for the presence or levels of A-beta using an antibodydescribed herein.

In another embodiment, more than one antibody or immunogen isadministered.

In another embodiment, the antibody, immunoconjugate, immunogen,composition or nucleic acid or vector is administered directly to thebrain or other portion of the CNS.

In another embodiment, the composition is a pharmaceutical compositioncomprising the compound or immunogen in admixture with apharmaceutically acceptable, diluent or carrier.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the disclosure aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the present disclosure will now be described inrelation to the drawings in which:

FIG. 1A: Schematic representations of cyclic peptides containing theepitope residues HHQK (SEQ ID NO: 1), including the cyclic peptideCGHHQKG (SEQ ID NO: 2) with circular peptide bond, the cyclic peptideC-PEG2-HHQKG (SEQ ID NO: 3) with PEG2 linker between the C and Hresidues, and the cyclic peptide CGHHQK-PEG2 (SEQ ID NO: 4) with PEG2linker between the K and C residues.

FIG. 1B: is a schematic showing a series of cyclic compounds comprisingQKLV (SEQ ID NO: 5).

FIG. 1C: Schematic representations of cyclic peptides comprising HDSG(SEQ ID NO: 9), including the cyclic peptide with circular peptide bond,the cyclic peptide with PEG2 linker between the G and C residues, andthe cyclic peptide with PEG2 linker between the C and H residues.

FIG. 2A: Immunohistochemical staining of plaque from cadaveric AD brainusing 6E10 positive control antibody.

FIG. 2B: Immunohistochemical staining of plaque from cadaveric AD brainusing an antibody (303-25-1B4) raised against cyclo(CGHDSGG) (SEQ IDNO:10).

FIG. 2C: Immunohistochemical staining of plaque from cadaveric AD brainusing purified monoclonal antibody (301-17, 12G11) raised againstcyclo(CGHHQKG) (SEQ ID NO: 2).

FIG. 2D: Immunohistochemical staining of plaque from cadaveric AD brainusing purified antibody (305-62, 8H10) raised against cyclo(CGQKLVG)(SEQ ID NO: 6).

FIG. 3A-FIG. 3D: Are a series of plots showing propagation of A-betaaggregation in vitro in the presence or absence of representativeantibody raised using a cyclic peptide comprising HHQK (SEQ ID NO: 1) orQKLV (SEQ ID NO: 5). Plots showing propagation of A-beta aggregation invitro in the presence (stars) or absence (squares) of representativeantibodies. E and F are plots showing propagation of A-beta aggregationin vitro in the presence or absence of a representative antibody raisedusing a cyclic peptide comprising HDSG (SEQ ID NO:9) or QKLV (SEQ ID NO:5), respectively.

FIG. 4A-FIG. 4F: A series of plots showing the viability of rat primarycortical neurons exposed to toxic A-beta oligomers (AβO) in the presenceor absence of different molar ratios of a negative isotype control (A)or an antibody raised against cyclo(CGHHQKG) (SEQ ID NO: 2) (B), raisedagainst cyclo(CGQKLV) (SEQ ID NO: 6) (C) and (D), raised againstcyclo(CGHHQKG) (SEQ ID NO: 2)(E), raised against cyclo(CGHDSGG) (SEQ IDNO: 10) F. Controls include neurons cultured alone (CTRL), neuronsincubated with antibody without oligomers and neurons cultured with theneuroprotective humanin peptide (HNG) with or without oligomers.

FIG. 5A: A plot showing the viability of rat primary cortical neuronsexposed to toxic A-beta oligomers (AβO) in the presence or absence ofdifferent molar ratios of an antibody raised using a cyclic peptidecomprising HHQK (SEQ ID NO: 1) (A) and prepared under low endotoxinconditions. Controls include neurons cultured alone (CTRL), neuronsincubated with antibody without oligomers and neurons cultured with theneuroprotective humanin peptide (HNG) with or without oligomers.

FIG. 5B: A plot showing the viability of rat primary cortical neuronsexposed to toxic A-beta oligomers in the presence or absence ofdifferent molar ratios of recombinant 301-17 antibody (IgG1).

FIG. 6A: A plot showing that the loss of short term memory formationcaused by A-beta oligomer is inhibited by an antibody that is specificand/or selective for a conformational epitope presented in SEQ ID NO: 2.

FIG. 6B: A plot showing that the loss of short term memory formationcaused by A-beta oligomer is inhibited by an antibody that was raisedusing an immunogen comprising cyclo(CGQKLVG).

FIG. 6C: A plot showing that the loss of short term memory formationcaused by A-beta oligomer is inhibited by recombinant antibody 303-25antibody in a mouse IgG1 framework (HDSG epitope (SEQ ID NO: 9))(antibody 1). Antibody 2 is mouse IgG1 isotype control.

FIG. 6D: A plot showing that the loss of short term memory formationcaused by A-beta oligomer is inhibited by a recombinant antibody 301-17in a mouse IgG1 framework (HHQK epitope (SEQ ID NO: 1)) in a mouse IgG1framework.

FIG. 7A-FIG. 7C: A series of plots showing hippocampal PSD-95 levels(A), SNAP25 levels (B) and TNF-alpha levels (C) in an vivo assay usingantibody 305-61 (QKLV epitope SEQ ID NO: 5).

FIG. 7D-FIG. 7F: A series of plots showing hippocampal PSD-95 levels(D), SNAP25 levels (E) and TNF-alpha levels (F) in an vivo assay usingrecombinant antibody 303-25 antibody in a mouse IgG1 framework (HDSGepitope(SEQ ID NO: 9)). Antibody 2 is a mouse IgG1 isotype control.

FIG. 7G, FIG. 7H: A series of plots showing hippocampal PSD-95 levels(G) and SNAP25 levels (H) in an vivo assay using recombinant antibody301-17 (HHQK epitope (SEQ ID NO: 1)) in a mouse IgG1 framework at lowconcentration.

FIG. 8 is a dot blot image showing antibody binding to monomeric,oligomeric and fibrillary A-beta.

DETAILED DESCRIPTION OF THE DISCLOSURE

Provided herein are methods for using antibodies, immunotherapeuticcompositions and methods which target epitopes preferentially accessiblein toxic oligomeric species of A-beta, including oligomeric speciesassociated with Alzheimer's disease. A region in A-beta has beenidentified that may be specifically and/or selectively accessible toantibody binding in oligomeric species of A-beta.

Generation of oligomer-specific or oligomer selective antibodies wasaccomplished through the identification of targets on A-beta peptidethat are not present, or present to a lesser degree, on either themonomer and/or fibril. Oligomer-specific epitopes need not differ inprimary sequence from the corresponding segment in the monomer orfibril, however they would be conformationally distinct in the contextof the oligomer. That is, they would present a distinct conformation interms of backbone and/or side-chain orientation in the oligomer thatwould not be present (or would be unfavourable) in the monomer and/orfibril.

Antibodies raised to linear peptide regions tend not to be selective foroligomer, and thus bind to monomer as well.

As described herein, to develop antibodies that may be selective foroligomeric forms of A-beta, the inventors sought to identify regions ofA-beta sequence that are prone to disruption in the context of thefibril, and that may be exposed as well on the surface of the oligomer.

The inventors have identified region predicted to be prone to disruptionin the context of the fibril. The inventors designed cyclic compoundscomprising the identified target region to satisfy criteria of analternate conformation such as a different curvature profile vs residueindex, higher exposed surface area, and/or did not readily align by rootmean squared deviation (RMSD) to either the linear or fibril ensembles.HQK, HHQK (SEQ ID NO: 1), HQKL (SEQ ID NO: 202), QKLV (SEQ ID NO: 5) andHDSG (SEQ ID NO: 9) were identified as regions prone to disruption andcyclic compounds were designed comprising the identified target regions.

As shown in the Examples, an immunogen comprising the cyclic compoundsSEQ ID Nos: 2, 6 and 10 were used to produce monoclonal antibodies.Antibodies could be raised using a cyclic peptide comprising the targetregion, that selectively bound the cyclic peptide compared to a linearpeptide of the same sequence (e.g. corresponding linear sequence).Experimental results are described and identify epitope-specific andconformationally selective antibodies that bind synthetic oligomerselectively compared to synthetic monomers. Further staining of AD braintissue identified antibodies that show no or negligible plaque bindingand in vitro studies found that the antibodies inhibited Aβ oligomerpropagation and aggregation. Furthermore, antibody that selectivelybinds SEQ ID Nos: 2, 6 or 10 inhibits and/or prevents neurotoxicity andloss of memory formation induced by soluble A-beta oligomers in a mousemodel.

I. Definitions

As used herein, the term ‘A-beta’ may alternately be referred to as‘amyloid beta’, ‘amyloid β’, A-beta, A-beta or ‘Aβ’. Amyloid beta is apeptide of 36-43 amino acids and includes all wild-type and mutant formsof all species, particularly human A-beta. A-beta40 refers to the 40amino acid form; A-beta42 refers to the 42 amino acid form, etc. Theamino acid sequence of human wildtype A-beta42 is shown in SEQ ID NO:19.

As used herein, the term “A-beta monomer” herein refers to any of theindividual subunit forms of the A-beta (e.g. 1-40, 1-42, 1-43) peptide.

As used herein, the term “A-beta oligomer” herein refers to a pluralityof any of the A-beta subunits wherein several (e.g. at least two) A-betamonomers are non-covalently aggregated in a conformationally-flexible,partially-ordered, three-dimensional globule of less than about 100, ormore typically less than about 50 monomers. For example, an oligomer maycontain 3 or 4 or 5 or more monomers. The term “A-beta oligomer” as usedherein includes both synthetic A-beta oligomer and/or native A-betaoligomer. “Native A-beta oligomer” refers to A-beta oligomer formed invivo, for example in the brain and CSF of a subject with AD.

As used herein, the term “A-beta fibril” refers to a molecular structurethat comprises assemblies of non-covalently associated, individualA-beta peptides which show fibrillary structure under an electronmicroscope. The fibrillary structure is typically a “cross beta”structure; there is no theoretical upper limit on the size of multimers,and fibrils may comprise thousands or many thousands of monomers.Fibrils can aggregate by the thousands to form senile plaques, one ofthe primary pathological morphologies diagnostic of AD.

The term “HHQK” means the amino acid sequence histidine, histidine,glutamine, lysine, as shown in SEQ ID NO: 1. Similarly HQK, HHQ, HHQKLV(SEQ ID NO: 8) HQKL (SEQ ID NO: 202) refers to the amino acid sequenceidentified by the 1-letter amino acid code. The term “QKLV” means theamino acid sequence glutamine, lysine, leucine, and valine, as shown inSEQ ID NO: 5 and “HDSG” means the amino acid sequence of histidine,aspartic acid, serine and glycine as shown in SEQ ID NO: 9. Depending onthe context, the reference of the amino acid sequence can refer to asequence in A-beta or an isolated peptide, such as the amino acidsequence of a cyclic compound.

The term “amino acid” includes all of the naturally occurring aminoacids as well as modified L-amino acids. The atoms of the amino acid caninclude different isotopes. For example, the amino acids can comprisedeuterium substituted for hydrogen nitrogen-15 substituted fornitrogen-14, and carbon-13 substituted for carbon-12 and other similarchanges.

The term “antibody” as used herein is intended to include, monoclonalantibodies, polyclonal antibodies, single chain, veneered, humanized andother chimeric antibodies and binding fragments thereof, including forexample a single chain Fab fragment, Fab′2 fragment or single chain Fvfragment. The antibody may be from recombinant sources and/or producedin animals such as rabbits, llamas, sharks etc. Also included are humanantibodies that can be produced in transgenic animals or usingbiochemical techniques or can be isolated from a library such as a phagelibrary. Humanized or other chimeric antibodies may include sequencesfrom one or more than one isotype or class or species.

The phrase “isolated antibody” refers to antibody produced in vivo or invitro that has been removed from the source that produced the antibody,for example, an animal, hybridoma or other cell line (such asrecombinant insect, yeast or bacteria cells that produce antibody). Theisolated antibody is optionally “purified”, which means at least: 80%,85%, 90%, 95%, 98% or 99% purity.

The term “binding fragment” as used herein to a part or portion of anantibody or antibody chain comprising fewer amino acid residues than anintact or complete antibody or antibody chain and which binds theantigen or competes with intact antibody. Exemplary binding fragmentsinclude without limitations Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv,dimers, nanobodies, minibodies, diabodies, and multimers thereof.Fragments can be obtained via chemical or enzymatic treatment of anintact or complete antibody or antibody chain. Fragments can also beobtained by recombinant means. For example, F(ab′)2 fragments can begenerated by treating the antibody with pepsin. The resulting F(ab′)2fragment can be treated to reduce disulfide bridges to produce Fab′fragments. Papain digestion can lead to the formation of Fab fragments.Fab, Fab′ and F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies,diabodies, bispecific antibody fragments and other fragments can also beconstructed by recombinant expression techniques.

The terms “IMGT numbering” or “ImMunoGeneTics database numbering”, whichare recognized in the art, refer to a system of numbering amino acidresidues which are more variable (i.e. hypervariable) than other aminoacid residues in the heavy and light chain variable regions of anantibody, or antigen binding portion thereof.

When an antibody is said to bind to an epitope within specifiedresidues, such as HHQK (SEQ ID NO: 1), what is meant is that theantibody specifically binds to a peptide or polypeptide containing thespecified residues or a part thereof for example at least 1 residue orat least 2 residues, with a minimum affinity, and does not bind anunrelated sequence or unrelated sequence spatial orientation greaterthan for example an isotype control antibody. Such an antibody does notnecessarily contact each residue of HHQK (SEQ ID NO: 1) (or a relatedepitope), and every single amino acid substitution or deletion withinsaid epitope does not necessarily significantly affect and/or equallyaffect binding affinity.

When an antibody is said to selectively bind an epitope such as aconformational epitope, such as HHQK (SEQ ID NO: 1), what is meant isthat the antibody preferentially binds one or more particularconformations containing the specified residues or a part thereof withgreater affinity than it binds said residues in another conformation.For example, when an antibody is said to selectively bind a cyclopeptidecomprising SEQ ID NO: 1, 5 or 9 or related epitope relative to acorresponding linear peptide, the antibody binds the cyclopeptide withat least a 2 fold greater affinity than it binds the linear peptide.

As used herein, the term “conformational epitope” refers to an epitopewhere the epitope amino acid sequence has a particular three-dimensionalstructure wherein at least an aspect of the three-dimensional structurenot present or less likely to be present in a corresponding linearpeptide is specifically and/or selectively recognized by the cognateantibody. The epitope e.g. HHQK (SEQ ID NO: 1), QKLV (SEQ ID NO: 5) orHDSG (SEQ ID NO: 9) may be partially or completely exposed on themolecular surface of oligomeric A-beta and partially or completelyobscured from antibody recognition in monomeric or fibrillar plaqueA-beta. Antibodies which specifically bind a conformation-specificepitope recognize the spatial arrangement of one or more of the aminoacids of that conformation-specific epitope. For example, an HHQK (SEQID NO: 1) conformational epitope refers to an epitope of HHQK (SEQ IDNO: 1) that is recognized by antibodies selectively, for example atleast 2 fold, 3 fold, 5 fold, 10 fold, 50 fold, 100 fold, 250 fold, 500fold or 1000 fold or greater more selectivity as compared to antibodiesraised using linear HHQK (SEQ ID NO: 1).

The term “related epitope” as used herein means at least two residues ofHHQK (SEQ ID NO: 1), QKLV (SEQ ID NO: 5) or HDSG (SEQ ID NO: 9) that areantigenic when for example conjugated to KLH, optionally with respect toHHQK sequences comprising HQK, and/or sequences comprising 1 2 or 3amino acid residues in a A-beta N-terminal and/or 1 residue C-terminalto at least two residues of HHQK (SEQ ID NO: 1), for HDSG (SEQ ID NO: 9)1 2 or 3 amino acid residues in a A-beta N-terminal and/or 1 2 or 3amino acid residues in a A-beta C-terminal to HDSG (SEQ ID NO: 9) or forQKLV (SEQ ID NO: 5), comprising 1 2 or 3 amino acid residues in a A-betaC-terminal and/or 1 residue N-terminal to QKLV (SEQ ID NO: 5). Forexample HHQK (SEQ ID NO: 1) and HQKL (SEQ ID NO: 202) which share thesubregion HQK were identified as regions prone to disorder in an A-betafibril. HQK and HQKL are accordingly related epitopes. Exemplary relatedepitopes can include epitopes whose sequences are shown in Table 8 (1).The related epitope is for example up to 6 A-beta residues.

The term “no or negligible plaque binding” or “lacks or has negligibleplaque binding” as used herein with respect to an antibody means thatthe antibody does not show typical plaque morphology staining onimmunohistochemistry (e.g. in situ) and the level of staining iscomparable to or no more than 2 fold the level seen with an IgG negative(e.g. irrelevant) isotype control.

The term “Isolated peptide” refers to peptide that has been produced,for example, by recombinant or synthetic techniques, and removed fromthe source that produced the peptide, such as recombinant cells orresidual peptide synthesis reactants. The isolated peptide is optionally“purified”, which means at least: 80%, 85%, 90%, 95%, 98% or 99% purityand optionally pharmaceutical grade purity.

The term “detectable label” as used herein refers to moieties such aspeptide sequences (such a myc tag, HA-tag, V5-tag or NE-tag),fluorescent proteins that can be appended or introduced into a peptideor compound described herein and which is capable of producing, eitherdirectly or indirectly, a detectable signal. For example, the label maybe radio-opaque, positron-emitting radionuclide (for example for use inPET imaging), or a radioisotope, such as ³H, ¹³N, ¹⁴C, ¹⁸F, ³²P, ³⁵S,¹²³I, ¹¹²I, ¹¹³I; a fluorescent (fluorophore) or chemiluminescent(chromophore) compound, such as fluorescein isothiocyanate, rhodamine orluciferin; an enzyme, such as alkaline phosphatase, beta-galactosidaseor horseradish peroxidase; an imaging agent; or a metal ion. Thedetectable label may be also detectable indirectly for example usingsecondary antibody.

The term “epitope” as commonly used means an antibody binding site,typically a polypeptide segment, in an antigen that is specificallyrecognized by the antibody. As used herein “epitope” can also refer tothe amino acid sequences or part thereof identified on A-beta using thecollective coordinates method described. For example an antibodygenerated against an isolated peptide corresponding to a cyclic compoundcomprising the identified target region HHQK SEQ ID NO: 1), recognizespart or all of said epitope sequence. An epitope is “accessible” in thecontext of the present specification when it is accessible to binding byan antibody.

The term “greater affinity” as used herein refers to a relative degreeof antibody binding where an antibody X binds to target Y more strongly(K_(on)) and/or with a smaller dissociation constant (K_(off)) than totarget Z, and in this context antibody X has a greater affinity fortarget Y than for Z. Likewise, the term “lesser affinity” herein refersto a degree of antibody binding where an antibody X binds to target Yless strongly and/or with a larger dissociation constant than to targetZ, and in this context antibody X has a lesser affinity for target Ythan for Z. The affinity of binding between an antibody and its targetantigen, can be expressed as K_(A) equal to 1/K_(D) where K_(D) is equalto k_(on)/k_(off). The k_(on) and k_(off) values can be measured usingsurface plasmon resonance technology, for example using a MolecularAffinity Screening System (MASS-1) (Sierra Sensors GmbH, Hamburg,Germany). An antibody that is selective for a conformation presented ina cyclic compound optional a cyclic peptide for example has a greateraffinity for the cyclic compound (e.g. cyclic peptide) compared to acorresponding sequence in linear form (e.g. the sequence non-cyclized).

Also as used herein, the term “immunogenic” refers to substances thatelicit the production of antibodies, activate T-cells and other reactiveimmune cells directed against an antigenic portion of the immunogen.

The term “corresponding linear compound” with regard to a cycliccompound refers to a compound, optionally a peptide, comprising orconsisting of the same sequence or chemical moieties as the cycliccompound but in linear (i.e. non-cyclized) form, for example havingproperties as would be present in solution of a linear peptide. Forexample, the corresponding linear compound can be the synthesizedpeptide that is not cyclized.

As used herein “specifically binds” in reference to an antibody meansthat the antibody recognizes an epitope sequence and binds to its targetantigen with a minimum affinity. For example a multivalent antibodybinds its target with a K_(D) of at least 1e−6, at least 1e−7, at least1e−8, at least 1e−9, or at least 1e−10. Affinities greater than at least1e−8 may be preferred. An antigen binding fragment such as Fab fragmentcomprising one variable domain, may bind its target with a 10 fold or100 fold less affinity than a multivalent interaction with anon-fragmented antibody.

The term “selectively binds” as used herein with respect to an antibodythat selectively binds a form of A-beta (e.g. fibril, monomer oroligomer) or a cyclic compound means that the antibody binds the formwith at least 2 fold, at least 3 fold, or at least 5 fold, at least 10fold, at least 100 fold, at least 250 fold, at least 500 fold or atleast 1000 fold or more greater affinity. Accordingly an antibody thatis more selective for a particular conformation (e.g. oligomer)preferentially binds the particular form of A-beta with at least 2 foldetc. greater affinity compared to another form and/or a linear peptide.

The term “linker” as used herein means a chemical moiety that can becovalently linked to the peptide comprising HHQK (SEQ ID NO: 1),optionally linked to HHQK (SEQ ID NO: 1) peptide N- and C-termini toproduce a cyclic compound. The linker can comprise a spacer and/or oneor more functionalizable moieties. The linker can be linked via thefunctionalizable moieties to a carrier protein or an immunogen enhancingagent such as keyhole limpet hemocyanin (KLH).

The term “spacer” as used herein means any preferably non-immunogenic orpoorly immunogenic chemical moiety that can be covalently-linkeddirectly or indirectly to a peptide N- and C-termini to produce a cycliccompound of longer length than the peptide itself, for example thespacer can be linked to the N- and C-termini of a peptide consisting ofHHQK (SEQ ID NO: 1) to produce a cyclic compound of longer backbonelength than the HHQK (SEQ ID NO: 1) sequence itself. That is, whencyclized the peptide with a spacer (for example of 3 amino acidresidues) makes a larger closed circle than the peptide without aspacer. The spacer may include, but is not limited to, non-immunogenicmoieties such as G, A, or PEG repeats. The spacer may comprise or becoupled to one or more functionalizing moieties, such as one or morecysteine (C) residues, which can be interspersed within the spacer orcovalently linked to one or both ends of the spacer. Where afunctionalizable moiety such as a C residue is covalently linked to oneor more termini of the spacer, the spacer is indirectly covalentlylinked to the peptide. The spacer can also comprise the functionalizablemoiety in a spacer residue as in the case where a biotin molecule isintroduced into an amino acid residue.

The term “functionalizable moiety” as used herein refers to a chemicalentity with a “functional group” which as used herein refers to a groupof atoms or a single atom that will react with another group of atoms ora single atom (so called “complementary functional group”) to form achemical interaction between the two groups or atoms. In the case ofcysteine, the functional group can be +SH which can be reacted to form adisulfide bond. Accordingly the linker can for example be CCC. Thereaction with another group of atoms can be covalent or a strongnon-covalent bond, for example as in the case as biotin-streptavidinbonds, which can have Kd˜1e−14. A strong non-covalent bond as usedherein means an interaction with a Kd of at least 1e−9, at least 1e−10,at least 1e−11, at least 1e−12, at least 1e−13 or at least 1e−14.

Proteins and/or other agents may be functionalized (e.g. coupled) to thecyclic compound, either to aid in immunogenicity, or to act as a probein in vitro studies. For this purpose, any functionalizable moietycapable of reacting (e.g. making a covalent or non-covalent but strongbond) may be used. In one specific embodiment, the functionalizablemoiety is a cysteine residue which is reacted to form a disulfide bondwith an unpaired cysteine on a protein of interest, which can be, forexample, an immunogenicity enhancing agent such as Keyhole limpethemocyanin (KLH), or a carrier protein such as Bovine serum albumin(BSA) used for in vitro immunoblots or immunohistochemical assays.

The term “reacts with” as used herein generally means that there is aflow of electrons or a transfer of electrostatic charge resulting in theformation of a chemical interaction.

The term “animal” or “subject” as used herein includes all members ofthe animal kingdom including mammals, optionally including or excludinghumans.

A “conservative amino acid substitution” as used herein, is one in whichone amino acid residue is replaced with another amino acid residuewithout abolishing the protein's desired properties. Suitableconservative amino acid substitutions can be made by substituting aminoacids with similar hydrophobicity, polarity, and R-chain length for oneanother. Examples of conservative amino acid substitution include:

Conservative Substitutions Type of Amino Acid Substitutable Amino AcidsHydrophilic Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr Sulphydryl CysAliphatic Val, Ile, Leu, Met Basic Lys, Arg, His Aromatic Phe, Tyr, Trp

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences or two nucleic acidsequences. To determine the percent identity of two amino acid sequencesor of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino acid or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions.times.100%). In one embodiment, thetwo sequences are the same length. The determination of percent identitybetween two sequences can also be accomplished using a mathematicalalgorithm. A preferred, non-limiting example of a mathematical algorithmutilized for the comparison of two sequences is the algorithm of Karlinand Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modifiedas in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A.90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLASTnucleotide searches can be performed with the NBLAST nucleotide programparameters set, e.g., for score=100, word length=12 to obtain nucleotidesequences homologous to a nucleic acid molecules of the presentapplication. BLAST protein searches can be performed with the XBLASTprogram parameters set, e.g., to score−50, word length=3 to obtain aminoacid sequences homologous to a protein molecule described herein. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. Alternatively, PSI-BLAST can be used to perform aniterated search which detects distant relationships between molecules(Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., of XBLAST andNBLAST) can be used (see, e.g., the NCBI website). Another preferrednon-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. The percent identity between twosequences can be determined using techniques similar to those describedabove, with or without allowing gaps. In calculating percent identity,typically only exact matches are counted.

For antibodies, percentage sequence identities can be determined whenantibody sequences maximally aligned by IMGT or other (e.g. Kabatnumbering convention). After alignment, if a subject antibody region(e.g., the entire mature variable region of a heavy or light chain) isbeing compared with the same region of a reference antibody, thepercentage sequence identity between the subject and reference antibodyregions is the number of positions occupied by the same amino acid inboth the subject and reference antibody region divided by the totalnumber of aligned positions of the two regions, with gaps not counted,multiplied by 100 to convert to percentage.

The term “nucleic acid sequence” as used herein refers to a sequence ofnucleoside or nucleotide monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof. The nucleic acid sequences of the presentapplication may be deoxyribonucleic acid sequences (DNA) or ribonucleicacid sequences (RNA) and may include naturally occurring bases includingadenine, guanine, cytosine, thymidine and uracil. The sequences may alsocontain modified bases. Examples of such modified bases include aza anddeaza adenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine. The nucleic acid can be either double stranded or singlestranded, and represents the sense or antisense strand. Further, theterm “nucleic acid” includes the complementary nucleic acid sequences aswell as codon optimized or synonymous codon equivalents. The term“isolated nucleic acid sequences” as used herein refers to a nucleicacid substantially free of cellular material or culture medium whenproduced by recombinant DNA techniques, or chemical precursors, or otherchemicals when chemically synthesized. An isolated nucleic acid is alsosubstantially free of sequences which naturally flank the nucleic acid(i.e. sequences located at the 5′ and 3′ ends of the nucleic acid) fromwhich the nucleic acid is derived.

“Operatively linked” is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid. Suitable regulatory sequences may be derived from avariety of sources, including bacterial, fungal, viral, mammalian, orinsect genes. Selection of appropriate regulatory sequences is dependenton the host cell chosen and may be readily accomplished by one ofordinary skill in the art. Examples of such regulatory sequencesinclude: a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other sequences, such as an origin of replication,additional DNA restriction sites, enhancers, and sequences conferringinducibility of transcription may be incorporated into the expressionvector.

The term “vector” as used herein comprises any intermediary vehicle fora nucleic acid molecule which enables said nucleic acid molecule, forexample, to be introduced into prokaryotic and/or eukaryotic cellsand/or integrated into a genome, and include plasmids, phagemids,bacteriophages or viral vectors such as retroviral based vectors, AdenoAssociated viral vectors and the like. The term “plasmid” as used hereingenerally refers to a construct of extrachromosomal genetic material,usually a circular DNA duplex, which can replicate independently ofchromosomal DNA.

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected which promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41(% (G+C)−600/l), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule a1% mismatch may be assumed to result in about a 1° C. decrease in Tm,for example if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In preferredembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5×Denhardt's solution/1.0% SDS at Tm−5° C. based on the aboveequation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3×SSC at42° C. It is understood, however, that equivalent stringencies may beachieved using alternative buffers, salts and temperatures. Additionalguidance regarding hybridization conditions may be found in: CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in:Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Laboratory Press, 2001.

The term “treating” or “treatment” as used herein and as is wellunderstood in the art, means an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disease, stabilized (i.e. not worsening) state of disease,preventing spread of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, diminishment of thereoccurrence of disease, and remission (whether partial or total),whether detectable or undetectable. “Treating” and “Treatment” can alsomean prolonging survival as compared to expected survival if notreceiving treatment. “Treating” and “treatment” as used herein alsoinclude prophylactic treatment. For example, a subject with early stageAD can be treated to prevent progression can be treated with a compound,antibody, immunogen, nucleic acid or composition described herein toprevent progression.

The term “administered” as used herein means administration of atherapeutically effective dose of a compound or composition of thedisclosure to a cell or subject.

As used herein, the phrase “effective amount” means an amount effective,at dosages and for periods of time necessary to achieve a desiredresult. Effective amounts when administered to a subject may varyaccording to factors such as the disease state, age, sex, weight of thesubject. Dosage regime may be adjusted to provide the optimumtherapeutic response.

The term “pharmaceutically acceptable” means that the carrier, diluent,or excipient is compatible with the other components of the formulationand not substantially deleterious to the recipient thereof.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” an antibody maycontain the antibody alone or in combination with other ingredients.

In understanding the scope of the present disclosure, the term“consisting” and its derivatives, as used herein, are intended to beclose ended terms that specify the presence of stated features,elements, components, groups, integers, and/or steps, and also excludethe presence of other unstated features, elements, components, groups,integers and/or steps.

The recitation of numerical ranges by endpoints herein includes allnumbers and fractions subsumed within that range (e.g. 1 to 5 includes1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood thatall numbers and fractions thereof are presumed to be modified by theterm “about.” Further, it is to be understood that “a,” “an,” and “the”include plural referents unless the content clearly dictates otherwise.The term “about” means plus or minus 0.1 to 50%, 5-50%, or 10-40%,preferably 10-20%, more preferably 10% or 15%, of the number to whichreference is being made.

Further, the definitions and embodiments described in particularsections are intended to be applicable to other embodiments hereindescribed for which they are suitable as would be understood by a personskilled in the art. For example, in the following passages, differentaspects of the invention are defined in more detail. Each aspect sodefined may be combined with any other aspect or aspects unless clearlyindicated to the contrary. In particular, any feature indicated as beingpreferred or advantageous may be combined with any other feature orfeatures indicated as being preferred or advantageous.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” can include a pluralityof compounds, including mixtures thereof.

II. Methods

Conformation selective antibodies were raised to A-beta epitopesequences presented in a cyclic format as described herein. Saidantibodies selectively bound synthetic and/or native oligomeric A-betaspecies compared to monomeric A-beta and A-beta fibril plaques. Furthersaid antibodies were able to inhibit in vitro propagation of A-betaaggregation. In addition, as demonstrated in toxicity assays, saidantibodies inhibited A-beta oligomer in vitro neural cell toxicity andprevented neurotoxicity and loss of memory formation induced by solubleA-beta oligomers in an in vivo mouse model. Several of the specificantibody sequences are described in PCT/CA2016/051300, filed Nov. 9,2016; PCT/CA2016/051305, filed Nov. 9, 2016; PCT/CA2016/051303, filedNov. 9, 2016; and PCT/CA2017/050866, filed Jul. 18, 2017. Additionalantibodies are described herein. Uses of said antibodies are alsoprovided.

III. Methods Using “Epitope” Compounds

Accordingly, the present disclosure provides for treating or preventinga disease or condition associated with and/or induced by soluble A-betaoligomer comprising administering to a subject in need thereof acompound, immunogen, composition, antibody, nucleic acid or celldescribed herein described herein.

In an embodiment, the compound, immunogen or antibody is directed to aconformational epitope in A-beta consisting of amino acids HHQK (SEQ IDNO: 1), HQKL (SEQ ID NO: 202) or a part thereof such as HQK, HHQK (SEQID NO: 1) corresponding to amino acids residues 13-16 on A-beta and HQKL(SEQ ID NO: 20) corresponding to amino acids 14-17. HHQK (SEQ ID NO: 1)and HQKL (SEQ ID NO: 20) were identified as regions prone to disorder inan A-beta fibril. The residues HHQK (SEQ ID NO: 1) and HQKL (SEQ ID NO:202) emerged in predictions.

The residues QKLV (SEQ ID NO: 5) were identified as a region prone todisorder in an A-beta fibril. In an embodiment, the compound, immunogenor antibody is directed to a conformational epitope in A-beta consistingof amino acids QKLV (SEQ ID NO: 5), a part thereof such as QKL or arelated epitope.

Also, the residues HDSG (SEQ ID NO: 9) were identified as a region proneto disorder in an A-beta fibril. In another embodiment, the compound,immunogen or antibody is directed to a conformational epitope in A-betaconsisting of amino acids HDSG (SEQ ID NO: 9), a part thereof such asHDS or a related epitope.

In another aspect the subject is administered a compound comprising anA-beta peptide comprising or consisting of QKLV (SEQ ID NO: 5), HDSG(SEQ ID NO:9), a part thereof or a related epitope sequence and orcombinations thereof.

In an aspect the subject is administered a compound comprising an A-betapeptide comprising or consisting of HHQK (SEQ ID NO: 1), a relatedepitope sequence including a part of any of the foregoing, wherein ifthe peptide is HHQK (SEQ ID NO: 1), the peptide is in a conformationthat is distinct in at least one feature from linear HHQK (SEQ ID NO:1). In an embodiment, the A-beta peptide is selected from HHQK (SEQ IDNO: 1). HQKL (SEQ ID NO: 202) and is comprised in a cyclic compound. Theepitopes are included in the epitopes collectively referred to herein asHHQK (SEQ ID NO: 1) and related epitopes (and their sequences arecollectively referred to as related epitope sequences). In anembodiment, the related epitope comprises or consists of HQKL (SEQ IDNO: 202), HQK and epitopes that comprise 1, 2 or 3 amino acids in A-betaeither N-terminal and/or 1 amino acid C-terminal to HQK.

In an embodiment, the A-beta peptide comprises HQK or HHQK (SEQ IDNO: 1) or a related epitope, can include 1, 2 or 3 additional residuesin A-beta N-terminus of and/or 1, 2 or 3 amino acid C-terminus of HHQK(SEQ ID NO: 1). For example, the 3 amino acids N-terminal to HHQK (SEQID NO: 1) in A-beta are YEV and the 3 amino acids C-terminal to HHQK(SEQ ID NO: 1) are LVF. In an embodiment, the A-beta peptide is amaximum of 7 or 6 A-beta residues. In an embodiment, the A-beta peptideis a maximum of 5 A-beta residues. In yet another embodiment A-betapeptide (e.g. in the compound such as a cyclic compound) is 4 A-betaresidues, optionally HHQK (SEQ ID NO: 1).

In an embodiment, the A-beta peptide comprising QKLV (SEQ ID NO: 5) caninclude 1, 2 or 3 additional residues in A-beta C-terminus and/or 1, 2or 3 additional residue in A-beta N-terminus of QKLV. The 3 amino acidsN-terminal to QKLV in A-beta are VHH and the 3 amino acids C-terminal toQKLV are FFA. In an embodiment the A-beta peptide is a maximum of 7amino acids, 6 amino acids or 5 amino acids.

In an embodiment, the A-beta peptide comprising HDSG (SEQ ID NO: 9) caninclude 1, 2 or 3 additional residues in A-beta C-terminus and/or 1, 2or 3 additional residue in A-beta N-terminus of HDSG. The 3 amino acidsN-terminal to HDSG in A-beta are EFR and the 3 amino acids C-terminal toHDSG are YEV. In an embodiment the A-beta peptide is a maximum of 7amino acids, 6 amino acids or 5 amino acids.

In an embodiment, the compound further includes a linker. The linkercomprises a spacer and/or one or more functionalizable moieties. Thelinker can for example comprise 1, 2, 3, 4, 5, 6, 7 or 8 amino acidsand/or equivalently functioning molecules such as polyethylene glycol(PEG) moieties, and/or a combination thereof. In an embodiment, thespacer amino acids are selected from non-immunogenic or poorlyimmunogenic amino acid residues such as G and A, for example the spacercan be GGG, GAG, G(PEG)G, PEG-PEG(also referred to as PEG2)-GG and thelike. One or more functionalizable moieties e.g. amino acids with afunctional group may be included for example for coupling the compoundto an agent or detectable tag or a carrier such as BSA or animmunogenicity enhancing agent such as KLH.

In an embodiment the linker comprises GC-PEG, PEG-GC, GCG or PEG2-CG.

In an embodiment, the linker comprises 1, 2, 3, 4, 5, 6, 7 or 8 aminoacids.

In certain embodiments, the cyclic compound has a maximum of 12, 11, 10,9, 8, or 7 residues, optionally amino acids and/or equivalent units suchas PEG units or other similar sized chemical moieties.

In embodiments wherein the A-beta peptide comprising for example HQK orHHQK (SEQ ID NO: 1), HDSG (SEQ ID NOL9) or QKLV (SEQ ID NO: 5), includes1, 2 or 3 additional residues found in A-beta that are N- and/orC-terminal to the epitope sequence the linker in the cyclized compoundis covalently linked to the N- and/or C-termini of the A-beta residues.Where the A-beta peptide is HHQK (SEQ ID NO: 1), the linker iscovalently linked to residues H and K.

In an embodiment, the compound is a cyclic compound, such as acyclopeptide, optionally a cyclopeptide described herein.

Proteinaceous portions of compounds (or the compound wherein the linkeris also proteinaceous) may be prepared by chemical synthesis usingtechniques well known in the chemistry of proteins such as solid phasesynthesis or synthesis in homogenous solution.

Reference to the “cyclic peptide” or “cyclopeptide” herein can refer toa fully proteinaceous compound (e.g. wherein the linker is for example1, 2, 3, 4, 5, 6, 7 or 8 amino acids). It is understood that propertiesdescribed for the cyclic peptide determined in the examples can beincorporated in other compounds (e.g. other cyclic compounds) comprisingnon-amino acid linker molecules.

The linear peptide comprising the A-beta sequence can be comprised in alinear compound. The linear compound or the linear peptide comprisingHHQK (SEQ ID NO: 1), QKLV (SEQ ID NO: 5) or HDSG (SEQ ID NO: 9) is in anembodiment, a corresponding linear peptide. In another embodiment, thelinear peptide is any length of A-beta peptide comprising the epitopesequence), including for example a linear peptide comprising A-betaresidues 1-35, or smaller portions thereof such as A-beta residues10-20, 11-20, 12-20, 13-20, 10-19, 10-18 and the like etc. The linearpeptide can in some embodiments also be a full length A-beta peptide.

The cyclic compound can be synthesized as a linear molecule with thelinker covalently attached to the N-terminus or C-terminus of thepeptide comprising the A-beta peptide, prior to cyclization.Alternatively part of the linker is covalently attached to theN-terminus and part is covalently attached to the C-terminus prior tocyclization. In either case, the linear compound is cyclized for examplein a head to tail cyclization (e.g. amide bond cyclization).

In some embodiments, the linker is indirectly coupled to the N- andC-terminus residues of the A-beta peptide.

In an embodiment, the cyclic compound is a compound in FIG. 1.

Methods for making cyclized peptides are known in the art and includeSS-cyclization or amide cyclization (head-to-tail, or backbonecyclization). Methods are further described in the Examples. Forexample, a peptide with “C” residues at its N- and C-termini, can bereacted by SS-cyclization to produce a cyclic peptide.

In an embodiment an immunogen comprising a compound, optionally a cycliccompound described herein is administered for treating or preventing anoligomeric A-beta disease. In an embodiment, the immunogen is preparedwith sterile reagents and/or distilled water.

In an embodiment, the immunogen is a cyclic peptide comprising A-betapeptide described herein.

In an embodiment, the immunogen comprises immunogenicity enhancing agentsuch as Keyhole Limpet Hemocyanin (KLH). The immunogenicity enhancingagent can be coupled to the compound either directly, such as through anamide bound, or indirectly through a chemical linker.

The immunogen can be produced by conjugating the cyclic compoundcontaining the A-beta peptide to an immunogenicity enhancing agent suchas Keyhole Limpet Hemocyanin (KLH) or a carrier such bovine serumalbumin (BSA) using for example the method described in Lateef et al2007, herein incorporated by reference. In an embodiment, the methoddescribed in Example 1 is used.

An immunogen is suitably prepared or formulated for administration to asubject, for example, the immunogen may be sterile, or purified.

A further aspect is administration of an isolated nucleic acid encodingthe proteinaceous portion of a compound or immunogen described herein.

In embodiment, the nucleic acid molecule encodes any one of the aminoacid sequences sent forth herein, such as antibody or fragment thereof,optionally a binding fragment.

A further aspect is administration of a vector comprising said nucleicacid. Suitable vectors are described elsewhere herein.

IV. Antibodies, Immunoconjugates, Cells and Nucleic Acids and UsesThereof

Also provided is an antibody or binding fragment described herein, cellsexpressing such antibody or binding fragment, such as hybridoma celllines as well as nucleic acids encoding said antibodies. Also providedare uses thereof for inhibiting A-beta propagation, and treating orpreventing A-beta oligomer associated and/or induced conditions anddiseases including memory loss, and Alzheimer's disease.

Accordingly, in an aspect, antibodies raised using the compounds andimmunogens, including compounds and immunogens comprising the cyclicpeptides described above can be used for treating AD and/or other A-betaamyloid associated and/or induced diseases and conditions.

Accordingly, an aspect includes an antibody or binding fragment thereofdescribed herein. In an embodiment the antibody or binding fragmentthereof comprises a light chain variable region and a heavy chainvariable region, optionally fused, the heavy chain variable regioncomprising complementarity determining regions CDR-H1, CDR-H2 andCDR-H3, the light chain variable region comprising complementaritydetermining region CDR-L1, CDR-L2 and CDR-L3 and with the amino acidsequences of said CDRs comprising the sequences in Table 9, optionallyTable 9B, 9C, 9G, 9H or 9I.

Also provided in another aspect is an immunoconjugate comprising anantibody or binding fragment described herein, optionally with the aminoacid sequences of said CDRs comprising the sequences in Table 9B, 9C,9G, 9H or 9I and a detectable label or cytotoxic agent, optionally,wherein the detectable label comprises a positron emitting radionuclide,optionally for use in subject imaging such as PET imaging.

A further aspect includes a hybrdioma cell line deposited under theprovisions of the Budapest Treaty with the American Type CultureCollection (ATCC®) 10801 University Blvd., Manassas, Va., 20110-2209,USA on Jul. 19, 2017 and given the Accession number PTA-124318.

Also provided in another aspect is an antibody produced by the hybrdiomacell line deposited under the provisions of the Budapest Treaty with theAmerican Type Culture Collection (ATCC®) 10801 University Blvd.,Manassas, Va., 20110-2209, USA on Jul. 19, 2017 and given the Accessionnumber PTA-124318.

Antibodies described herein and immunoconjugates comprising a cytotoxicagent can be used to inhibit A-beta oligomer associated and/or inducedconditions and diseases and/or to inhibit A-beta oligomer propagation.

Accordingly, an aspect includes a method of treating or preventing adisease or condition associated with and/or induced by soluble A-betaoligomer comprising administering to a subject in need thereof: anisolated conformation specific and/or selective antibody or bindingfragment thereof that specifically and/or selectively binds to a cycliccompound comprising an A-beta peptide having a sequence of QKL, HQK,KLV, HHQK (SEQ ID NO: 1), QKLV (SEQ ID NO: 5) or HDSG (SEQ ID NO: 9),they cyclic compound optionally having a sequence of SEQ ID NO: 2, 3, 4,6, 7, 8, 10, 11 or 12; an immunogen comprising a cyclic compoundcomprising an A-beta peptide having a sequence of QKL, HQK, KLV, HHQK(SEQ ID NO: 1), QKLV (SEQ ID NO: 5) or HDSG (SEQ ID NO: 9); a cellexpressing said antibody or binding fragment thereof; or a nucleic acidencoding said antibody or binding fragment thereof.

Also provided in an embodiment, is a method of inhibiting A-betaoligomer propagation, the method comprising contacting a cell or tissueexpressing A-beta with or administering to a subject in need thereof aneffective amount of an isolated A-beta oligomer specific or selectiveantibody or binding fragment thereof that specifically and/orselectively binds to a cyclic compound comprising an A-beta peptidehaving a sequence of QKL, HQK, KLV, HHQK (SEQ ID NO: 1), QKLV (SEQ IDNO: 5) or HDSG (SEQ ID NO: 9), they cyclic compound optionally having asequence of SEQ ID NO: 2, 3, 4, 6, 7, 8, 10, 11 or 12; an immunogencomprising a cyclic compound comprising an A-beta peptide having asequence of QKL, HQK, KLV, HHQK (SEQ ID NO: 1), QKLV (SEQ ID NO: 5) orHDSG (SEQ ID NO: 9); a cell expressing said antibody or binding fragmentthereof; or a nucleic acid encoding said antibody or binding fragmenttherefore immunoconjugate thereof, to inhibit A-beta aggregation and/oroligomer propagation.

In an embodiment, the method includes administration of an antibody(including a binding fragment thereof) that specifically binds to anA-beta peptide having a sequence HQK, HHQK (SEQ ID NO: 1), QKLV (SEQ IDNO: 5), HDSG (SEQ ID NO: 9) or a related epitope sequence describedherein.

In an embodiment the antibody is specific and/or selective for A-betapeptide presented in the cyclic compound.

In an embodiment, the cyclic compound is a cyclic peptide optionally onedescribed herein. The terms cyclopeptide and cyclic peptide are usedinterchangeably herein.

In an embodiment, the antibody specifically and/or selectively binds theA-beta peptide presented in the cyclic compound relative to acorresponding linear compound comprising the A-beta peptide.

In an embodiment, the antibody does not bind a linear peptide comprisingthe sequence HHQK (SEQ ID NO: 1), QKLV (SEQ ID NO: 5), or HDSG (SEQ IDNO: 9) optionally wherein the sequence of the linear peptide is a linearversion of a cyclic sequence used to raise the antibody, optionally asset forth in SEQ ID NOs: 2, 6 or 10.

In an embodiment the antibody is isolated. In an embodiment, theantibody is an exogenous antibody.

In an embodiment, the antibody does not specifically bind and/or is notselective for linear HQKLVF (SEQ ID NO: 17), linear HQKLVFF (SEQ ID NO:18), linear HQKLVFFAED (SEQ ID NO: 13), or linear HHQKLVFFAEDVGSNK (SEQID NO: 14) relative to cyclic compound comprising an A-beta peptideconsisting of HHQK (SEQ ID NO: 1), HQK or HQKL (SEQ ID NO: 202). In anembodiment, the antibody does not specifically bind and/or is notselective for linear peptides consisting of HHQK (SEQ ID NO: 1).Selective binding can be measured using an ELISA or surface plasmonresonance measurement, as described herein.

In an embodiment, the antibody does not bind a linear peptide comprisingthe sequence QKLV (SEQ ID NO: 5) or HDSG (SEQ ID NO: 9), optionallywherein the sequence of the linear peptide is a linear version of acyclic sequence used to raise the antibody as described herein.

In an embodiment, the antibody does not specifically or appreciably bindmonomeric A-beta. In an embodiment, the antibody does not specificallyor appreciably bind A-beta senile plaques, for example, in situ in ADbrain tissue.

In another embodiment, the antibody does not selectively bind monomericA-beta compared to native- or synthetic-oligomeric A-beta.

In an embodiment, the antibody selectively binds a cyclic compoundcomprising SEQ ID NO: 1 or a part thereof, SEQ ID NO: 5 or a partthereof, or SEQ ID NO: 9 or a part thereof or a related epitopeoptionally in the context of cyclo(CGHHQKG) (SEQ ID NO: 2),cyclo(CGQKLVG) (SEQ ID NO: 6) or cyclo (CGHDSGG) (SEQ ID NO: 10),respectively relative to the corresponding linear peptide. For example,in an embodiment the antibody selectively binds HHQK (SEQ ID NO: 1) QKLV(SEQ ID NO: 5), HDSG (SEQ ID NO: 9) or a related epitope sequence in acyclic conformation and has at least 2 fold, at least 5 fold, at least10 fold at least 20 fold, at least 30 fold, at least 40 fold, at least50 fold, at least 100 fold, at least 500 fold, at least 1000 fold moreselectivity for the epitope in the cyclic conformation compared to alinear compound such as a corresponding linear compound, for example asmeasured by ELISA or surface plasmon resonance, optionally using amethod described herein.

In an embodiment, the antibody selectively binds a cyclic compoundcomprising the epitope sequence relative to linear peptide or a speciesof A-beta such as A-beta oligomer relative to monomer. In an embodiment,the selectivity is at least 2 fold, at least 3 fold, at least 5 fold, atleast 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, atleast 50 fold, at least 100 fold, at least 500 fold, at least 1000 foldmore selective for the cyclic compound and/or A-beta oligomer over aspecies of A-beta selected from A-beta monomer and/or A-beta fibril

In an embodiment, the A-beta oligomer comprises A-beta 1-42 subunits.

In an embodiment, the antibody lacks A-beta fibril plaque (also referredto as senile plaque) staining. Absence of plaque staining can beassessed by comparing to a positive control such as A-beta-specificantibodies 6E10 and 4G8 (Biolegend, San Diego, Calif.), or 2C8 (EnzoLife Sciences Inc., Farmingdale, N.Y.) and an isotype control. Anantibody described herein lacks or has negligible A-beta fibril plaquestaining if the antibody does not show typical plaque morphologystaining and the level of staining is comparable to or no more than 2fold the level seen with an IgG negative isotype control. The scale canfor example set the level of staining with isotype control at 1 and with6E10 at 10. An antibody lacks A-beta fibril plaque staining if the levelof staining on such a scale is 2 or less.

In embodiment, the antibody shows minimal A-beta fibril plaque staining,for example on the foregoing scale, levels scored at less about or lessthan 3.

In an embodiment, the antibody is produced using a cyclic compound orimmunogen described herein, optionally using a method described herein.

In an embodiment, the antibody is a monoclonal antibody. In anembodiment, the antibody is a chimeric antibody such as a humanizedantibody comprising the CDR sequences as recited in Table 9.

To produce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from a subject immunized with an immunogen describedherein, and fused with myeloma cells by standard somatic cell fusionprocedures thus immortalizing these cells and yielding hybridoma cells.Such techniques are well known in the art, (e.g. the hybridoma techniqueoriginally developed by Kohler and Milstein (Nature 256:495-497 (1975))as well as other techniques such as the human B-cell hybridoma technique(Kozbor et al., Immunol. Today 4:72 (1983)), the EBV-hybridoma techniqueto produce human monoclonal antibodies (Cole et al., Methods Enzymol,121: 140-67 (1986)), and screening of combinatorial antibody libraries(Huse et al., Science 246:1275 (1989)). Hybridoma cells can be screenedimmunochemically for production of antibodies specifically reactive withthe desired epitopes and the monoclonal antibodies can be isolated.

Specific antibodies, or antibody fragments, reactive against particularantigens or molecules, may also be generated by screening expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria with cell surface components. For example, complete Fabfragments, VH regions and FV regions can be expressed in bacteria usingphage expression libraries (see for example Ward et al., Nature41:544-546 (1989); Huse et al., Science 246:1275-1281 (1989); andMcCafferty et al., Nature 348:552-554 (1990).

In an embodiment, the antibody is a humanized antibody.

The humanization of antibodies from non-human species has been welldescribed in the literature. See for example EP-B1 0 239400 and Carter &Merchant 1997 (Curr Opin Biotechnol 8, 449-454, 1997 incorporated byreference in their entirety herein). Humanized antibodies are alsoreadily obtained commercially (eg. Scotgen Limited, 2 Holly Road,Twickenham, Middlesex, Great Britain.).

Humanized forms of rodent antibodies are readily generated by CDRgrafting (Riechmann et al. Nature, 332:323-327, 1988). In this approachthe six CDR loops comprising the antigen binding site of the rodentmonoclonal antibody are linked to corresponding human framework regions.CDR grafting often yields antibodies with reduced affinity as the aminoacids of the framework regions may influence antigen recognition (Foote& Winter. J Mol Biol, 224: 487-499, 1992). To maintain the affinity ofthe antibody, it is often necessary to replace certain frameworkresidues by site directed mutagenesis or other recombinant techniquesand may be aided by computer modeling of the antigen binding site (Co etal. J Immunol, 152: 2968-2976, 1994).

Humanized forms of antibodies are optionally obtained by resurfacing(Pedersen et al. J Mol Biol, 235: 959-973, 1994). In this approach onlythe surface residues of a rodent antibody are humanized.

Humanized antibodies can be produced as antigen binding fragments suchas Fab, Fab′ F(ab′)2, Fd, Fv and single domain antibody fragments, or assingle chain antibodies in which the heavy and light chains are linkedby a spacer. Also, the human or humanized antibodies may exist inmonomeric or polymeric form. The humanized antibody optionally comprisesone non-human chain and one humanized chain (i.e. one humanized heavy orlight chain).

In an embodiment, the humanized antibody comprises a heavy chainvariable domain and/or light chain variable domain listed in Table 12 or13 or a sequence with at least 50% or more sequence identity theretowherein the CDR sequences are maintained.

Human antibodies specific to a particular antigen may be identified by aphage display strategy (Jespers et al. Bio/Technology, 12: 899-903,1994). In one approach, the heavy chain of a rodent antibody directedagainst a specific antigen is cloned and paired with a repertoire ofhuman light chains for display as Fab fragments on filamentous phage.The phage is selected by binding to antigen. The selected human lightchain is subsequently paired with a repertoire of human heavy chains fordisplay on phage, and the phage is again selected by binding to antigen.The result is a human antibody Fab fragment specific to a particularantigen. In another approach, libraries of phage are produced wheremembers display different human antibody fragments (Fab or Fv) on theirouter surfaces (Dower et al., WO 91/17271 and McCafferty et al., WO92/01047). Phage displaying antibodies with a desired specificity areselected by affinity enrichment to a specific antigen. The human Fab orFv fragment identified from either approach may be recloned forexpression as a human antibody in mammalian cells.

Human antibodies are optionally obtained from transgenic animals (U.S.Pat. Nos. 6,150,584; 6,114,598; and 5,770,429). In this approach theheavy chain joining region (JH) gene in a chimeric or germ-line mutantmouse is deleted. Human germ-line immunoglobulin gene array issubsequently transferred to such mutant mice. The resulting transgenicmouse is then capable of generating a full repertoire of humanantibodies upon antigen challenge.

Antibodies including humanized or human antibodies are selected from anyclass of immunoglobulins including: IgM, IgG, IgD, IgA or IgE; and anyisotype, including: IgG1, IgG2, IgG3 and IgG4. The humanized or humanantibody may include sequences from one or more than one isotype orclass.

Additionally, antibodies specific for the epitopes described herein arereadily isolated by screening antibody phage display libraries. Forexample, an antibody phage library is optionally screened by using adisease specific epitope of the current invention to identify antibodyfragments specific for the disease specific epitope. Antibody fragmentsidentified are optionally used to produce a variety of recombinantantibodies that are useful with different embodiments of the presentinvention. Antibody phage display libraries are commercially available,for example, through Xoma (Berkeley, Calif.) Methods for screeningantibody phage libraries are well known in the art.

A further aspect is administration of an antibody and/or bindingfragment thereof comprising a light chain variable region and a heavychain variable region, the heavy chain variable region comprisingcomplementarity determining regions CDR-H1, CDR-H2 and CDR-H3, the lightchain variable region comprising complementarity determining regionCDR-L1, CDR-L2 and CDR-L3 and with the amino acid sequences of said CDRscomprising the sequences set forth in Table 9.

In an embodiment, the antibody is a humanized antibody comprising theCDR sequences as recited in Table 9.

Also provided in another embodiment, is an antibody or binding fragmentthereof or administration of an antibody or binding fragment thereofcomprising the CDRs in Table 9 or Table 15 and a light chain variableregion and a heavy chain variable region, optionally in the context of asingle chain antibody.

For example, the antibody and/or binding fragment thereof administeredcomprises a light chain variable region and a heavy chain variableregion, the heavy chain variable region comprising complementaritydetermining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variableregion comprising complementarity determining region CDR-L1, CDR-L2 andCDR-L3 and with the amino acid sequences of said CDRs comprising thesequences set forth in Table 9.

In an embodiment, the antibody or binding fragment comprises a heavychain variable region comprising: i) an amino acid sequence of a heavychain variable sequence as set forth in Table 10; ii) an amino acidsequence with at least 50%, at least 60%, at least 70%, at least 80% orat least 90% sequence identity to said heavy chain variable sequence setout in Table 10, 12 or 13, wherein the CDR sequences are thecorresponding CDRs as set forth in Table 9, or iii) a conservativelysubstituted amino acid sequence i) wherein the CDR sequences are thecorresponding CDRs as set forth in Table 9; and wherein the antibody orbinding fragment thereof comprises a light chain variable regioncomprising i) an amino acid sequence of a light chain variable sequenceas set forth in Table 10, 12 or 13, ii) an amino acid sequence with atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%sequence identity to said light chain variable sequence as set out inTable 10, 12 or 13, wherein the CDR sequences are the corresponding CDRsas set forth in Table 9, or iii) a conservatively substituted amino acidsequence i) wherein the CDR sequences a are the corresponding CDRs asset forth in Table 9.

For example, in an embodiment the antibody administered comprises aheavy chain variable region comprises: i) an amino acid sequence as setforth in SEQ ID NO: 84; ii) an amino acid sequence with at least 50%, atleast 60%, at least 70%, at least 80% or at least 90% sequence identityto SEQ ID NO: 84, wherein the CDR sequences are as set forth in SEQ IDNO: 20, 21 and 22, or iii) a conservatively substituted amino acidsequence i). In another aspect the antibody administered comprises alight chain variable region comprising i) an amino acid sequence as setforth in SEQ ID NO: 86, ii) an amino acid sequence with at least 50%, atleast 60%, at least 70%, at least 80% or at least 90% sequence identityto SEQ ID NO: 86, wherein the CDR sequences are as set forth in SEQ IDNO: 23, 24 and 25 or iii) a conservatively substituted amino acidsequence of i), wherein the CDR sequences are as set forth in SEQ ID NO:23, 24 and 25. In another embodiment, the heavy chain variable regionamino acid sequence is encoded by a nucleotide sequence as set out inSEQ ID NO: 83 or a codon degenerate optimized version thereof. Inanother embodiment, the antibody comprises a light chain variable regionamino acid sequence encoded by a nucleotide sequence as set out in SEQID NO: 85 or a codon degenerate or optimized version thereof. In anembodiment, the heavy chain variable region comprises an amino acidsequence as set forth in SEQ ID NO: 84. In an embodiment, the lightchain variable region comprises an amino acid sequence as set forth inSEQ ID NO: 86.

As another example, the antibody comprises a heavy chain variable regioncomprises: i) an amino acid sequence as set forth in SEQ ID NO: 98 or102; ii) an amino acid sequence with at least 50%, at least 60%, atleast 70%, at least 80% sequence identity to SEQ ID NO: 98 or 102,wherein the CDR sequences are as set forth in SEQ ID NO: 41, 42, 43, 47,48, and/or 49, or iii) a conservatively substituted amino acid sequencei) wherein the CDR sequences are as set forth in SEQ ID NO:41, 42, 43,47, 48, and/or 49. In another aspect the antibody comprises a lightchain variable region comprising i) an amino acid sequence as set forthin SEQ ID NO: 100 or 104, ii) an amino acid sequence with at least 50%,at least 60%, at least 70%, at least 80% 70% sequence identity to SEQ IDNO:100 or 104, wherein the CDR sequences are as set forth in SEQ ID NO:44, 45, 46, 50, 51, and/or 52, or iii) a conservatively substitutedamino acid sequence of i) wherein the CDR sequences are as set forth inSEQ ID NO: 41, 42, 43, 47, 48, and/or 49. In another embodiment, theheavy chain variable region amino acid sequence is encoded by anucleotide sequence as set out in SEQ ID NO: 97 or 101 or a codondegenerate optimized version thereof. In another embodiment, theantibody comprises a light chain variable region amino acid sequenceencoded by a nucleotide sequence as set out in SEQ ID NO: 99 or 103 or acodon degenerate or optimized version thereof. In an embodiment, theheavy chain variable region comprises an amino acid sequence as setforth in SEQ ID NO: 98 or 102. In an embodiment, the light chainvariable region comprises an amino acid sequence as set forth in SEQ IDNO: 100 or 104.

Other similar examples there the variable region of the heavy and/orlight chain have at least 50%, at least 60%, at least 70%, at least 80%70% sequence identity and where the CDRs are maintained can bedetermined on the basis of the Tables 9 and 10, 12 and 14.

In another aspect the antibody that is administered is an antibody thatspecifically binds a same epitope as the antibody with CDR sequences asrecited in Table 9, with variable regions as recited in Table 10, 12 or13 or produced by the hybrdioma cell line deposited under the provisionsof the Budapest Treaty with the American Type Culture Collection (ATCC®)10801 University Blvd., Manassas, Va., 20110-2209, USA on Jul. 19, 2017and given the Accession number PTA-124318.

Another aspect is an antibody that specifically binds A-beta or SEQ IDNO: 2, 6 or 10 (e.g. the same epitope as the antibody with CDR sequencesas recited in Table 9).

Another aspect includes an antibody or binding fragment oradministration of an antibody that competes for binding to human A-betawith an antibody comprising the CDR sequences as recited in Table 9 or15, or the variable domain sequences in Tables 10, 12 or 13.

In an embodiment, the antibody or binding fragment thereof competes forbinding with an antibody comprising a light chain variable region and aheavy chain variable region the heavy chain variable region comprisingcomplementarity determining regions CDR-H1, CDR-H2 and CDR-H3, the lightchain variable region comprising complementarity determining regionCDR-L1, CDR-L2 and CDR-L3 and with the amino acid sequences of said CDRscomprising the sequences in Tables 9B, 9C, 9G, 9H or 9I.

Competition between antibodies can be determined for example using anassay in which an antibody under test is assessed for its ability toinhibit specific binding of a reference antibody to the common antigen.A test antibody competes with a reference antibody if an excess of atest antibody (e.g., at least a 2 fold, 5, fold, 10 fold or 20 fold)inhibits binding of the reference antibody by at least 50%, at least75%, at least 80%, at least 90% or at least 95% as measured in acompetitive binding assay.

In a further aspect the antibody administered is an antibody conjugatedto a therapeutic, detectable label or cytotoxic agent.

A further aspect relates to an antibody complex comprising an antibodydescribed herein and/or a binding fragment thereof and oligomericA-beta.

In a further aspect an isolated nucleic acid encoding an antibody orpart thereof described herein is administered.

Nucleic acids encoding a heavy chain or a light chain can also beadministered, for example encoding a heavy chain comprising CDR-H1,CDR-H2 and/or CDR-H3 regions described herein or encoding a light chaincomprising CDR-L1, CDR-L2 and/or CDR-L3 regions described herein.

The present disclosure also includes administration of variants of thenucleic acid sequences that encode for the antibody and/or bindingfragment thereof disclosed herein. For example, the variants includenucleotide sequences that hybridize to the nucleic acid sequencesencoding the antibody and/or binding fragment thereof disclosed hereinunder at least moderately stringent hybridization conditions or codondegenerate or optimized sequences. In another embodiment, the variantnucleic acid sequences have at least 50%, at least 60%, at least 70%,most preferably at least 80%, even more preferably at least 90% and evenmost preferably at least 95% sequence identity to nucleic acid sequencesencoding in Table 10, 12 or 13.

A further aspect is an isolated nucleic acid encoding an antibodydescribed herein. In embodiments said isolated nucleic acid isadministered to a subject in need thereof.

Another aspect is an expression cassette or vector comprising thenucleic acid herein disclosed. In an embodiment, the vector is anisolated vector, optionally for administration to a subject in needthereof.

The vector can be any vector, including vectors suitable for producingan antibody and/or binding fragment thereof or expressing a peptidesequence described herein.

The nucleic acid molecules may be incorporated in a known manner into anappropriate expression vector which ensures expression of the protein.Possible expression vectors include but are not limited to cosmids,plasmids, or modified viruses (e.g. replication defective retroviruses,adenoviruses and adeno-associated viruses). The vector should becompatible with the host cell used. The expression vectors are “suitablefor transformation of a host cell”, which means that the expressionvectors contain a nucleic acid molecule encoding the peptidescorresponding to epitopes or antibodies described herein.

In an embodiment, the vector is suitable for expressing for examplesingle chain antibodies by gene therapy. The vector can be adapted forspecific expression in neural tissue, for example using neural specificpromoters and the like. In an embodiment, the vector comprises an IRESand allows for expression of a light chain variable region and a heavychain variable region. Such vectors can be used to deliver antibody invivo.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes.

Examples of such regulatory sequences include: a transcriptionalpromoter and enhancer or RNA polymerase binding sequence, a ribosomalbinding sequence, including a translation initiation signal.Additionally, depending on the host cell chosen and the vector employed,other sequences, such as an origin of replication, additional DNArestriction sites, enhancers, and sequences conferring inducibility oftranscription may be incorporated into the expression vector.

In an embodiment, the regulatory sequences direct or increase expressionin neural tissue and/or cells.

In an embodiment, the vector is a viral vector.

The recombinant expression vectors may also contain a marker gene whichfacilitates the selection of host cells transformed, infected ortransfected with a vector for expressing an antibody or epitope peptidedescribed herein.

The recombinant expression vectors may also contain expression cassetteswhich encode a fusion moiety (i.e. a “fusion protein”) which providesincreased expression or stability of the recombinant peptide; increasedsolubility of the recombinant peptide; and aid in the purification ofthe target recombinant peptide by acting as a ligand in affinitypurification, including for example tags and labels described herein.Further, a proteolytic cleavage site may be added to the targetrecombinant protein to allow separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein.Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne,Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to therecombinant protein.

Systems for the transfer of genes for example into neurons and neuraltissue both in vitro and in vivo include vectors based on viruses, mostnotably Herpes Simplex Virus, Adenovirus, Adeno-associated virus (AAV)and retroviruses including lentiviruses. Alternative approaches for genedelivery include the use of naked, plasmid DNA as well as liposome-DNAcomplexes. Another approach is the use of AAV plasmids in which the DNAis polycation-condensed and lipid entrapped and introduced into thebrain by intracerebral gene delivery (Leone et al. US Application No.2002076394).

Accordingly, in another aspect, the compounds, immunogens, nucleicacids, vectors and antibodies described herein for administration may beformulated in vesicles such as liposomes, nanoparticles, and viralprotein particles, for example for delivery of antibodies, compounds,immunogens and nucleic acids described herein. In particular syntheticpolymer vesicles, including polymersomes, can be used to administerantibodies.

Also provided in another aspect is administration of a cell, optionallyan isolated and/or recombinant cell, expressing an antibody describedherein or comprising a vector herein disclosed.

The recombinant cell can be generated using any cell suitable forproducing a polypeptide, for example suitable for producing an antibodyand/or binding fragment thereof. For example to introduce a nucleic acid(e.g. a vector) into a cell, the cell may be transfected, transformed orinfected, depending upon the vector employed.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the proteins described herein may be expressedin bacterial cells such as E. coli, insect cells (using baculovirus),yeast cells or mammalian cells.

In an embodiment, the cell is a eukaryotic cell selected from a yeast,plant, worm, insect, avian, fish, reptile and mammalian cell.

In an embodiment, the cell is a neural cell.

Yeast and fungi host cells suitable for expressing an antibody orpeptide include, but are not limited to Saccharomyces cerevisiae,Schizosaccharomyces pombe, the genera Pichia or Kluyveromyces andvarious species of the genus Aspergillus. Examples of vectors forexpression in yeast S. cerivisiae include pYepSec1, pMFa, pJRY88, andpYES2 (Invitrogen Corporation, San Diego, Calif.). Protocols for thetransformation of yeast and fungi are well known to those of ordinaryskill in the art.

Mammalian cells that may be suitable include, among others: COS (e.g.,ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No.CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573) and NS-1cells. Suitable expression vectors for directing expression in mammaliancells generally include a promoter (e.g., derived from viral materialsuch as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), aswell as other transcriptional and translational control sequences.Examples of mammalian expression vectors include pCDM8 and pMT2PC.

In an embodiment, the cell is a fused cell producing an antibodyspecific and/or selective for an epitope or epitope sequence describedherein, including for example that selectively binds A-beta oligomersover A-beta monomers, selectively binds an epitope sequence presented ina cyclic compound relative to a linear compound or lacks or hasnegligible plaque binding.

V. Compositions and Methods for Using

A further aspect is a composition comprising a compound, immunogen,nucleic acid, vector or antibody or binding fragment described herein.In embodiments, said composition is administered in a method describedherein.

The composition can for example include 1, 2, 3 or more antibodies orbinding fragments thereof, immunoconjugates, compounds, cells or nucleicacids described herein.

In an embodiment, the composition comprises a diluent.

Suitable diluents for nucleic acids include but are not limited towater, saline solutions and ethanol.

Suitable diluents for polypeptides, including antibodies or fragmentsthereof and/or cells include but are not limited to saline solutions, pHbuffered solutions and glycerol solutions or other solutions suitablefor freezing polypeptides and/or cells.

In an embodiment, the composition is a pharmaceutical compositioncomprising any of the peptides, immunogens, antibodies, nucleic acids orvectors disclosed herein, and optionally comprising a pharmaceuticallyacceptable carrier.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionsthat can be administered to subjects, optionally as a vaccine, such thatan effective quantity of the active substance is combined in a mixturewith a pharmaceutically acceptable vehicle.

Pharmaceutical compositions include, without limitation, lyophilizedpowders or aqueous or non-aqueous sterile injectable solutions orsuspensions, which may further contain antioxidants, buffers,bacteriostats and solutes that render the compositions substantiallycompatible with the tissues or the blood of an intended recipient. Othercomponents that may be present in such compositions include water,surfactants (such as Tween), alcohols, polyols, glycerin and vegetableoils, for example. Extemporaneous injection solutions and suspensionsmay be prepared from sterile powders, granules, tablets, or concentratedsolutions or suspensions. The composition may be supplied, for examplebut not by way of limitation, as a lyophilized powder which isreconstituted with sterile water or saline prior to administration tothe patient.

Pharmaceutical compositions may comprise a pharmaceutically acceptablecarrier. Suitable pharmaceutically acceptable carriers includeessentially chemically inert and nontoxic compositions that do notinterfere with the effectiveness of the biological activity of thepharmaceutical composition. Examples of suitable pharmaceutical carriersinclude, but are not limited to, water, saline solutions, glycerolsolutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammoniumchloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), andliposomes. Such compositions should contain a therapeutically effectiveamount of the compound, together with a suitable amount of carrier so asto provide the form for direct administration to the patient.

The composition may be in the form of a pharmaceutically acceptable saltwhich includes, without limitation, those formed with free amino groupssuch as those derived from hydrochloric, phosphoric, acetic, oxalic,tartaric acids, etc., and those formed with free carboxyl groups such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylarnino ethanol,histidine, procaine, etc.

In an embodiment comprising a compound or immunogen described herein,the composition comprises an adjuvant.

Adjuvants that can be used for example, include Intrinsic adjuvants(such as lipopolysaccharides) normally are the components of killed orattenuated bacteria used as vaccines. Extrinsic adjuvants areimmunomodulators which are typically non-covalently linked to antigensand are formulated to enhance the host immune responses. Aluminumhydroxide, aluminum sulfate and aluminum phosphate (collectivelycommonly referred to as alum) are routinely used as adjuvants. A widerange of extrinsic adjuvants can provoke potent immune responses toimmunogens. These include saponins such as Stimulons (Q521, Aquila,Worcester, Mass.) or particles generated therefrom such as ISCOMs and(immunostimulating complexes) and ISCOMATRIX, complexed to membraneprotein antigens (immune stimulating complexes), pluronic polymers withmineral oil, killed mycobacteria and mineral oil, Freund's completeadjuvant, bacterial products such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

In an embodiment, the adjuvant is aluminum hydroxide. In anotherembodiment, the adjuvant is aluminum phosphate. Oil in water emulsionsinclude squalene; peanut oil; MF59 (WO 90/14387); SAF (SyntexLaboratories, Palo Alto, Calif.); and Ribi™ (Ribi Immunochem, Hamilton,Mont.). Oil in water emulsions may be used with immunostimulating agentssuch as muramyl peptides (for example,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MOP),N-acetylnnurannyl-L-alanyl-D-isoglutannyl-L-alanine-2-(1′-2′dipamitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide(TM)), or other bacterial cell wallcomponents.

The adjuvant may be administered with an immunogen as a singlecomposition. Alternatively, an adjuvant may be administered before,concurrent and/or after administration of the immunogen.

Commonly, adjuvants are used as a 0.05 to 1.0 percent solution inphosphate-buffered saline. Adjuvants enhance the immunogenicity of animmunogen but are not necessarily immunogenic themselves. Adjuvants mayact by retaining the immunogen locally near the site of administrationto produce a depot effect facilitating a slow, sustained release ofimmunogen to cells of the immune system. Adjuvants can also attractcells of the immune system to an immunogen depot and stimulate suchcells to elicit immune responses. As such, embodiments may encompasscompositions further comprising adjuvants.

Adjuvants for parenteral immunization include aluminum compounds (suchas aluminum hydroxide, aluminum phosphate, and aluminum hydroxyphosphate). The antigen can be precipitated with, or adsorbed onto, thealuminum compound according to standard protocols. Other adjuvants suchas RIBI (ImmunoChem, Hamilton, Mont.) can also be used in parenteraladministration.

Adjuvants for mucosal immunization include bacterial toxins (e.g., thecholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridiumdifficile toxin A and the pertussis toxin (PT), or combinations,subunits, toxoids, or mutants thereof). For example, a purifiedpreparation of native cholera toxin subunit B (CTB) can be of use.Fragments, homologs, derivatives, and fusion to any of these toxins arealso suitable, provided that they retain adjuvant activity. Preferably,a mutant having reduced toxicity is used. Suitable mutants have beendescribed (e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627(Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PTmutant)). Additional LT mutants that can be used in the methods andcompositions include, for example Ser-63-Lys, Ala-69-Gly, Glu-110-Asp,and Glu-112-Asp mutants. Other adjuvants (such as a bacterialmonophosphoryl lipid A (MPLA) of various sources (e.g., E. coli,Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri,saponins, or polylactide glycolide (PLGA) microspheres) can also be usedin mucosal administration.

Other adjuvants include cytokines such as interleukins for example IL-1,IL-2 and IL-12, chemokines, for example CXCL10 and CCL5, macrophagestimulating factor, and/or tumor necrosis factor. Other adjuvants thatmay be used include CpG oligonucleotides (Davis. Curr Top MicrobiolImmunol., 247:171-183, 2000).

Oil in water emulsions include squalene; peanut oil; MF59 (WO 90/14387);SAF (Syntex Laboratories, Palo Alto, Calif.); and Ribi™ (RibiImmunochem, Hamilton, Mont.). Oil in water emulsions may be used withimmunostimulating agents such as muramyl peptides (for example,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylnnurannyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylnnurannyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide(TM)), or other bacterial cell wallcomponents.

Adjuvants useful for both mucosal and parenteral immunization includepolyphosphazene (for example, WO 95/2415), DC-chol (3b-(N—(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol (for example,U.S. Pat. No. 5,283,185 and WO 96/14831) and QS-21 (for example, WO88/9336).

An adjuvant may be coupled to an immunogen for administration. Forexample, a lipid such as palmitic acid, may be coupled directly to oneor more peptides such that the change in conformation of the peptidescomprising the immunogen does not affect the nature of the immuneresponse to the immunogen.

In an embodiment, the composition comprises an antibody describedherein. In another embodiment, the composition comprises an antibodydescribed herein and a diluent. In an embodiment, the composition is asterile composition.

A further aspect provides prior to administering the compound,immunogen, composition, antibody, nucleic acid or cell described hereindetecting whether a biological sample comprises A-beta oligomers themethod comprising contacting the biological sample with an antibodydescribed herein and/or detecting the presence of any antibody complex.In an embodiment the method is for detecting whether the biologic samplecomprises oligomeric A-beta.

In an embodiment, if A-beta oligomer is detected the subject isadministered a compound, immunogen, antibody or compositions describedherein.

In an embodiment, the method comprises:

a. contacting the biologic sample with an antibody described herein thatis specific and/or selective for A-beta oligomer herein under conditionspermissive to produce an antibody: A-beta oligomer complex;

b. detecting the presence of any complex;

wherein the presence of detectable complex is indicative that the samplemay contain A-beta oligomer; and

c. treating the subject with a compound, immunogen, antibody orcomposition described herein.

In an embodiment, the level of complex formed is compared to a testantibody such as a suitable Ig control or irrelevant antibody.

In an embodiment, the detection is quantitated and the amount of complexproduced is measured. The measurement can for example be relative to astandard.

In an embodiment, the measured amount is compared to a control.

In another embodiment, the method comprises:

(a) contacting a test sample of said subject with an antibody describedherein, under conditions permissive to produce an antibody-antigencomplex;

(b) measuring the amount of the antibody-antigen complex in the testsample; and

(c) comparing the amount of antibody-antigen complex in the test sampleto a control;

wherein detecting antibody-antigen complex in the test sample ascompared to the control indicates that the sample comprises oligomericA-beta and treating the subject with an antibody described herein ifsample comprises oligomeric A-beta above a threshold.

The control can be a sample control (e.g. from a subject without AD, orfrom a subject with a particular form of AD, mild, moderate oradvanced), or be a previous sample from the same subject for monitoringchanges in A-beta oligomer levels in the subject.

In an embodiment, an antibody described herein is used.

In an embodiment, the sample is a biological sample. In an embodiment,the sample comprises brain tissue or an extract thereof and/or CSF. Inan embodiment, the sample comprises whole blood, plasma or serum. In anembodiment, the sample is obtained from a human subject. In anembodiment, the subject is suspected of, at a risk of or has AD.

A number of methods can be used to detect an A-beta: antibody complexand thereby determine if the sample comprises A-beta oligomers arepresent in a sample using the antibodies described herein, includingimmunoassays such as flow cytometry, Western blots, ELISA, andimmunoprecipitation followed by SDS-PAGE immunocytochemistry.

As described in the Examples surface plasmon resonance technology can beused to assess conformation specific binding. If the antibody is labeledor a detectably labeled secondary antibody specific for the complexantibody is used, the label can be detected. Commonly used reagentsinclude fluorescent emitting and HRP labeled antibodies. In quantitativemethods, the amount of signal produced can be measured by comparison toa standard or control. The measurement can also be relative.

A further aspect includes a method of measuring a level of or imagingA-beta in a subject or tissue, optionally where the A-beta to bemeasured or imaged is oligomeric A-beta prior to administering, acompound, immunogen, composition, antibody, nucleic acid or celldescribed herein. In an embodiment, the method comprises administeringto a subject at risk or suspected of having or having AD, an antibodyconjugated to a detectable label; and detecting the label, optionallyquantitatively detecting the label. The label in an embodiment is apositron emitting radionuclide which can for example be used in PETimaging.

A further aspect includes a method of inducing an immune response in asubject to treat or prevent a disease associated with and/or induced bysoluble A-beta, comprising administering to the subject a compound,immunogen and/or composition comprising a compound described herein.

In an embodiment, the immunogen administered comprises a compound ofFIG. 1.

In an embodiment, the subject is a non-human subject such as a rodent.Antibody producing cells generated can also be used in an embodiment toproduce a hybridoma cell line.

It is demonstrated herein that the antibodies raised against immunogenscomprising SEQ ID NO: 2, 6 and 10, can specifically and/or selectivelybind A-beta oligomers and lack A-beta plaque staining. Oligomeric A-betaspecies are believed to be the toxic propagating species in AD. Furtheras shown herein, said antibodies inhibited A-beta aggregation and A-betaoligomer propagation. Accordingly, also provided are methods ofinhibiting A-beta oligomer propagation, the method comprising contactinga cell or tissue expressing A-beta with or administering to a subject inneed thereof an effective amount of an A-beta oligomer specific orselective antibody described herein to inhibit A-beta aggregation and/oroligomer propagation. In vitro the assay can be monitored as describedin the Examples.

The antibodies can be used for example for treating AD and/or otherA-beta amyloid associated and/or induced diseases and conditions. Forexample, variants of Lewy body dementia and in inclusion body myositis(a muscle disease) exhibit similar plaques as AD and A-beta can alsoform aggregates implicated in cerebral amyloid angiopathy.

In an embodiment the method is for treating or preventing AD and/orother A-beta amyloid associated and/or induced diseases or conditions,the method comprising administering to a subject in need thereof i) aneffective amount of an antibody described herein, optionally an A-betaoligomer specific or selective or a pharmaceutical compositioncomprising said antibody; or 2) administering an isolated cycliccompound comprising SEQ ID NO: 1 5 or 9 or a related epitope sequence orimmunogen or pharmaceutical composition comprising said cyclic compound,to a subject in need thereof. In other embodiments, nucleic acidsencoding the antibodies or immunogens described herein can also beadministered to the subject, optionally using vectors suitable fordelivering nucleic acids in a subject.

In an embodiment, a biological sample from the subject to be treated isassessed for the presence or levels of A-beta using an antibodydescribed herein. In an embodiment, a subject with detectable A-betalevels (e.g. A-beta antibody complexes measured in vitro or measured byimaging) is treated with the antibody.

The antibody and immunogens can for example be comprised in apharmaceutical composition as described herein, and formulated forexample in vesicles for improving delivery.

One or more antibodies targeting HHQK (SEQ ID NO: 1), HDSG (SEQ ID NO:9) or QKLV (SEQ ID NO: 5) and/or related antibodies can be administeredin combination. In addition the antibodies disclosed herein can beadministered with one or more other treatments such as a beta-secretaseinhibitor or a cholinesterase inhibitor.

In an embodiment, the antibody is a conformation specific/selectiveantibody, optionally that specifically or selectively binds A-betaoligomer.

Also provided are uses of the compositions, antibodies, isolatedpeptides, immunogens and nucleic acids for treating or preventing ADand/or other A-beta associated and/or induced diseases or conditions.

In an embodiment, the disease or condition associated with and/orinduced by soluble A-beta oligomer is cognitive deficits, optionallyloss of short term memory formation.

In an embodiment, the disease or condition associated with and/orinduced by soluble soluble A-beta oligomer A-beta is Alzheimer's disease(AD).

In an embodiment, the treatment is prophylactic treatment. For examplein an embodiment, the antibody is administered to a subject with apredisposition to developing a disease or condition associated withand/or induced by soluble A-beta oligomer.

In an embodiment, the disease or condition is perirhinal cortexdysfunction or pathology. In another embodiment, the disease orcondition is enthorinal cortex dysfunction or pathology.

In an embodiment, the disease or condition is associated with and/orinduced by soluble A-beta 1-42 oligomer.

The compositions, compounds, antibodies, isolated peptides, immunogensand nucleic acids, vectors etc. described herein can be administered forexample, by parenteral, intravenous, subcutaneous, intramuscular,intracranial, intraventricular, intrathecal, intraorbital, ophthalmic,intraspinal, intracisternal, intraperitoneal, intranasal, aerosol ororal administration.

In certain embodiments, the pharmaceutical composition is administeredsystemically.

In other embodiments, the pharmaceutical composition is administereddirectly to the brain or other portion of the CNS. For example suchmethods include the use of an implantable catheter and a pump, whichwould serve to discharge a pre-determined dose through the catheter tothe infusion site. A person skilled in the art would further recognizethat the catheter may be implanted by surgical techniques that permitvisualization of the catheter so as to position the catheter adjacent tothe desired site of administration or infusion in the brain. Suchtechniques are described in Elsberry et al. U.S. Pat. No. 5,814,014“Techniques of Treating Neurodegenerative Disorders by Brain Infusion”,which is herein incorporated by reference. Also contemplated are methodssuch as those described in US patent application 20060129126 (Kaplittand During “Infusion device and method for infusing material into thebrain of a patient”. Devices for delivering drugs to the brain and otherparts of the CNS are commercially available (eg. SynchroMed® EL InfusionSystem; Medtronic, Minneapolis, Minn.)

In another embodiment, the pharmaceutical composition is administered tothe brain using methods such as modifying the compounds to beadministered to allow receptor-mediated transport across the blood brainbarrier.

Other embodiments contemplate the co-administration of the compositions,compounds, antibodies, isolated peptides, immunogens and nucleic acidsdescribed herein with biologically active molecules known to facilitatethe transport across the blood brain barrier.

Also contemplated in certain embodiments, are methods for administeringthe compositions, compounds, antibodies, isolated peptides, immunogensand nucleic acids described herein across the blood brain barrier suchas those directed at transiently increasing the permeability of theblood brain barrier as described in U.S. Pat. No. 7,012,061 “Method forincreasing the permeability of the blood brain barrier”, hereinincorporated by reference.

VI. Kits for Use in Methods

A further aspect relates to a kit comprising i) an antibody and/orbinding fragment thereof, ii) a nucleic acid, iii) peptide or immunogen,iv) composition or v) recombinant cell described herein, comprised in avial such as a sterile vial or other housing and optionally a referenceagent and/or instructions for use thereof for use in treating orpreventing a disease or condition associated with and/or induced bysoluble A-beta oligomer.

In an embodiment, the kit further comprises one or more of a collectionvial, standard buffer and detection reagent.

The above disclosure generally describes the present application. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of theapplication. Changes in form and substitution of equivalents arecontemplated as circumstances might suggest or render expedient.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1

Immunogens

Cyclic peptides cyclo(CGHHQKG) (SEQ ID NO: 2) cyclo(CGQKLVG) (SEQ ID NO:6), and cyclo(CGHDSGG) (SEQ ID NO: 10) and corresponding linear peptideswere prepared (CPC Scientific, Sunnyvale, Calif., USA)

Peptides were generated at CPC Scientific, Sunnyvale, Calif., USA (bothcyclic and linear). Peptides were conjugated to KLH (for immunizing) andBSA (for screening) using a trifluoroacetate counter ion protocol.Peptides were desalted and checked by MS and HPLC and deemed 95% pure.Peptides were shipped to IPA for use in production of monoclonalantibodies in mouse.

Antibody Generation

Hybridomas and monoclonal antibodies were generated to cyclo(CGHHQKG)(SEQ ID NO: 2), cyclo(CGQKLVG) (SEQ ID NO: 6) and cyclo (CGHDSGG) (SEQID NO: 10) each linked to Keyhole Limpet Hemocyanin (KLH).

The cyclopeptides linked to KLH were sent for mouse monoclonal antibodyproduction (ImmunoPrecise Antibodies LTD (Victoria BC, Canada),following protocols approved by the Canadian Council on Animal Care.Mouse sera were screened using the conformational peptide used forproducing the antibodies linked to BSA and the linear peptides.

Fifty day old female BALB/c mice (Charles River Laboratories, Quebec)were immunized. A series of subcutaneous aqueous injections containingantigen but no adjuvant were given over a period of 19 days. Mice wereimmunized with 100 μg per mouse per injection of a 0.5 mg/mL cyclicpeptide-KLH solution in sterile saline. All 4 mice were euthanized onDay 19 and lymphocytes were harvested for hybridoma cell linegeneration.

Fusion/Hybridoma Development

Lymphocytes were isolated and fused with murine SP2/0 myeloma cells inthe presence of poly-ethylene glycol (PEG 1500). Fused cells werecultured using HAT selection. This method uses a semi-solidmethylcellulose-based HAT selective medium to combine the hybridomaselection and cloning into one step. Single cell-derived hybridomas growto form monoclonal colonies on the semi-solid media. 10 days after thefusion event, resulting hybridoma clones were transferred to 96-welltissue culture plates and grown in HT containing medium until mid-loggrowth was reached (5 days).

Example 2 Hybridoma Analysis

Tissue culture supernatants from the hybridomas were tested by indirectELISA on screening antigen (cyclic peptide-BSA) and probed for both IgGand IgM antibodies using a Goat anti-IgG/IgM(H&L)-HRP secondary anddeveloped with TMB substrate. Clones >0.2 OD in this assay were taken tothe next round of testing. Positive cultures were retested on screeningantigen to confirm secretion and on an irrelevant antigen (HumanTransferrin) to eliminate non-specific mAbs and rule out falsepositives. Selected clones were isotyped by antibody trapping ELISA todetermine if they are IgG or IgM isotype. Selected clones were alsotested by indirect ELISA on other cyclic peptide-BSA conjugates as wellas linear peptide-BSA conjugates to evaluate cross-reactivity and linkerreactivity. Antibodies were also screened by SPR analysis.

ELISA Antibody Screening

ELISA plates were coated with 1) 0.1 ug/well cyclopeptide-conjugated-BSAat 100 uL/well in carbonate coating buffer (pH 9.6) 0/N at 4C; 2) 0.1ug/well linear-peptide-conjugated-BSA at 100 uL/well in carbonatecoating buffer (pH 9.6) 0/N at 4C; or 3) 0.1 ug/well Negative-Peptide at100 uL/well in carbonate coating buffer (pH 9.6) 0/N at 4 C. PrimaryAntibody: Hybridoma supernatant at 100 uL/well incubated for 1 hour at37C with shaking. Secondary Antibody 1:10,000 Goat anti-mouseIgG/IgM(H+L)-HRP at 100 uL/well in PBS-Tween for 1 hour at 37C withshaking. All washing steps were performed for 30 mins with PBS-Tween.The substrate TMB was added at 50 uL/well, developed in the dark andstopped with equal volume 1M HCl.

SPR Binding Assays SPR Analysis of Antibody Binding to Cyclic Peptides,A-Beta Monomers and Oligomers

A-Beta Monomer and Oligomer Preparation:

Recombinant A-beta40 and 42 peptides (California Peptide, Salt Lake CityUtah, USA) were dissolved in ice-cold hexafluoroisopropanol (HFIP). TheHFIP was removed by evaporation overnight and dried in a Speed Vaccentrifuge. To prepare monomers, the peptide film was reconstituted inDMSO to 5 mM, diluted further to 100 μM in dH₂O and used immediately.Oligomers were prepared by diluting the 5 mM DMSO peptide solution inphenol red-free F12 medium (Life Technologies Inc., Burlington ON,Canada) to a final concentration of 100 μM and incubated for 24 hours to7 days at 4° C.

SPR Analysis of Cyclic Peptide, A-Beta Monomer and Oligomer Binding:

All SPR measurements were performed using a Molecular Affinity ScreeningSystem (MASS-1) (Sierra Sensors GmbH, Hamburg, Germany), an analyticalbiosensor that employs high intensity laser light and high speed opticalscanning to monitor binding interactions in real time. The primaryscreening of tissue culture supernatants was performed using an SPRdirect binding assay, whereby BSA-conjugated peptides, A-beta42 Monomerand A-beta42 Oligomer are covalently immobilized on individual flowcells of a High Amine Capacity (HAC) sensorchip (Sierra Sensors GmbH,Hamburg, Germany) and antibodies flowed over the surface. Each samplewas diluted and injected in duplicate over the immobilized peptide andBSA reference surfaces, followed by injection of running buffer only forthe dissociation phase. After every analytical cycle, the sensor chipsurfaces were regenerated. Sensorgrams were double-referenced bysubtracting out binding from the BSA reference surfaces and blankrunning buffer injections, and binding response report points collectedin the dissociation phase.

Protein G purified mAbs were analyzed in a secondary screen using an SPRindirect (capture) binding assay, whereby the antibodies wereimmobilized on a protein A-derivatized sensorchip (XanTec BioanalyticsGmbH, Duesseldorf, Germany) and A-beta40 Monomer, A-beta42 Oligomer,pooled soluble brain extracts flowed over the surface. The specificityof the antibodies was verified in an SPR direct binding assay bycovalently immobilizing A-beta42 Monomer and A-beta42 Oligomer onindividual flow cells of a HAC sensorchip and flowing purified mAbs overthe surface.

To further verify and validate A-beta42 Oligomer binding, antibodieswere covalently immobilized, followed by the injection over the surfaceof commercially-prepared stable A-beta42 Oligomers (SynAging SAS,Vandoeurve-iés-Nancy, France).

Isotyping

The hybridoma antibodies were isotyped using antibody trap experiments.Trap plates were coated with 1:10,000 Goat anti-mouse IgG/IgM(H&L)antibody at 100 uL/well carbonate coating buffer pH9.6 overnight at 4 C.No blocking step was used. Primary antibody (hybridoma supernatants) wasadded (100 ug/mL). Secondary Antibody 1:5,000 Goat anti-mouse IgGγ-HRPor 1:10,000 Goat anti-mouse IgMμ-HRP at 100 uL/well in PBS-Tween for 1hour at 37C with shaking. All washing steps were performed for 30 minswith PBS-Tween. The substrate TMB was added at 50 uL/well, developed inthe dark and stopped with equal volume 1M HCl.

Antibody Sequencing

The CDR and variable regions of the heavy and light chains weresequenced. Immunoglobulin gene transcripts expressed by the hybridomaswere amplified from cDNA generated from the hybridoma cells usingstandard RT-PCR and sequenced using a standard dye-terminator capillarysequencing method.

Biological Deposit

The 301-17 hybridoma was deposited under the provisions of the BudapestTreaty with the American Type Culture Collection (ATCC®) 10801University Blvd., Manassas, Va., 20110-2209, USA on Jul. 19, 2017 andgiven the Accession number PTA-124318.

Example 3 HHQK (SEQ ID NO: 1) Antibodies to Cyclo(CGHHQKG)

The antibodies were tested as described in Example 2.

ELISA and SPR testing found that hybridoma clones bound the cyclopeptidepreferentially over the linear peptide.

Clones 301-3, 301-11 and 301-17 raised against cyclo(CGHHQKG) wereselected for further analysis.

Isotyping revealed 301-3, 301-11 and 301-17 were IgG3 subtypes.

Antibodies were tested in one or more assays for their ability to bindcyclic peptide, linear peptide, A-beta 1-42 monomer and A-beta 1-42oligomers prepared as described above.

ELISA and SPR assays confirmed that the antibodies preferentially boundthe cylopeptide relative to the linear peptide (and were not crossreactive to unrelated cyclic peptides) and/or preferentially bound Aboligomers relative to monomers.

Antibody Sequence

Clones 301-3, 301-11 and 301-17 antibodies were sequenced as furtherprovided below. The CDR sequences of 301-3 and 301-11 are provided inTable 9(C and B). The CDRs for 301-17 are provided in SEQ ID NOs: 20-25.The consensus DNA sequence and polypeptide sequences of the variableportion of the heavy and light chain of the antibodies are provided inTable 10A.

As shown, the heavy chain CDRs for 301-3 and 301-11 were identical forCDRs 1 and 2 and CDR3 varied at one position. Two light chains weresequenced for 301-3. One light chain was near identical to the lightchain for 301-11.

Example 4 Antibodies to Cyclo(CGQKLVG)

The antibodies were tested as described in Example 2.

ELISA and SPR testing found that hybridoma clones bound the cyclopeptidepreferentially over the linear peptide.

Clones 305-59, 305-61, 350-62 raised against cyclo(CGQKLVG) wereselected for further analysis.

Isotyping revealed 305-59, 305-61, 350-62 were IgG1, IgG3 and IgG1subtypes respectively.

Antibodies were tested in one or more assays for their ability to bindcyclic peptide, linear peptide, A-beta 1-42 monomer and A-beta 1-42oligomers prepared as described above.

ELISA and SPR assays confirmed that the antibodies preferentially boundthe cylopeptide relative to the linear peptide (and were not crossreactive to unrelated cyclic peptides) and/or preferentially bound Aboligomers relative to monomers.

Antibody Sequence

Clones 305-59, 305-61, 350-62 antibodies were sequenced as furtherprovided below. The CDR sequences of 305-61, 301-62 and 305-59, areprovided in Table 9 (D, E and I). The consensus DNA sequence andpolypeptide sequences of the variable portion of the heavy and lightchain of the antibodies are provided in Table 10B.

Example 5 Antibodies to Cyclo(CGHDSGG) (SEQ ID NO: 10)

The antibodies were tested as described in Example 2.

ELISA and SPR testing found that hybridoma clones bound the cyclopeptidepreferentially over the linear peptide.

Clones 303-25, 303-26 and 303-30 raised against cyclo(CGHDSGG) wereselected for further analysis.

Isotyping revealed 303-25, 303-26 and 303-30 were IgG2a, IgG1 and IgG1subtypes respectively. Threes clones of antibody 303-26 and three clonesof 303-30 which differed in their kappa chain were identified.

Antibodies were tested in one or more assays for their ability to bindcyclic peptide, linear peptide, A-beta 1-42 monomer and A-beta 1-42oligomers prepared as described above.

ELISA and SPR assays confirmed that the antibodies preferentially boundthe cylopeptide relative to the linear peptide (and were not crossreactive to unrelated cyclic peptides) and/or preferentially bound Aboligomers relative to monomers.

Antibody Sequence

Clones 303-25, 303-26 and 303-30 antibodies were sequenced as furtherprovided below. The CDR sequences are provided in Table 9 (F, G and H).The consensus DNA sequence and polypeptide sequences of the variableportion of the heavy and light chain of the antibodies are provided inTable 10C.

Example 6 Methods Immunohistochemistry

Immunohistochemistry was performed on frozen human brain sections, withno fixation or antigen retrieval. In a humidified chamber, non-specificstaining was blocked by incubation with serum-free protein blockingreagent (Dako Canada Inc., Mississauga, ON, Canada) for 1 h. Thefollowing primary antibodies were used for immunostaining: mousemonoclonal isotype controls IgG1, IgG2a, and IgG2b, and anti-amyloidβ6E10, all purchased from Biolegend, and purified antibodies 301-11,301-17, 305-59, 305-61, 305-62, 303-25, 303-26 and 303-30 reactive tothe corresponding cyclopeptide. All antibodies were used at 1 μg/mL.Sections were incubated at room temperature for 1 h, and washed 3×5 minin TBS-T. Anti-Mouse IgG Horseradish Peroxidase conjugated (1:1000, ECL)was applied to sections and incubated 45 min, then washed 3×5 min inTBS-T. DAB chromogen reagent (Vector Laboratories, Burlington ON,Canada) was applied and sections rinsed with distilled water when thedesired level of target to background staining was achieved. Sectionswere counterstained with Mayer's haematoxylin, dehydrated and coverslips were applied. Slides were examined under a light microscope (ZeissAxiovert 200M, Carl Zeiss Canada, Toronto ON, Canada) and representativeimages captured at 20 and 40× magnification using a Leica DC300 digitalcamera and software (Leica Microsystems Canada Inc., Richmond Hill, ON).Images were optimized in Adobe Photoshop using Levels Auto Correction.

CSF and Brain Extracts Brain Extracts

Human brain tissues were obtained from the University of Maryland Brainand Tissue Bank upon approval from the UBC Clinical Research EthicsBoard (C04-0595). CSFs were obtained from patients assessed at the UBCHospital Clinic for Alzheimer's and Related Disorders. The study wasapproved by the UBC Clinical Research Ethics Board, and written consentfrom the participant or legal next of kin was obtained prior tocollection of CSF samples. Clinical diagnosis of probable AD was basedon NINCDS-ADRDA criteria. CSFs were collected in polypropylene tubes,processed, aliquoted into 100 μL polypropylene vials, and stored at −80°C. within 1 hour after lumbar puncture.

Homogenization:

Human brain tissue samples were weighed and subsequently submersed in avolume of fresh, ice cold TBS and EDTA-free protease inhibitor cocktailfrom Roche Diagnostics (Laval QC, Canada) such that the finalconcentration of brain tissue was 20% (w/v). Tissue was homogenized inthis buffer using a mechanical probe homogenizer (3×30 sec pulses with30 sec pauses in between, all performed on ice). TBS homogenized sampleswere then subjected to ultracentrifugation (70,000×g for 90 min).Supernatants were collected, aliquoted and stored at −80° C. The proteinconcentration of TBS homogenates was determined using a BCA proteinassay (Pierce Biotechnology Inc, Rockford Ill., USA).

CSF

CSF was pooled from 9 donors with AD and 9 donors without AD. Sampleswere analyzed by SPR using purified IgG at a concentration of 30micrograms/ml for all antibodies. Mouse IgG was used as an antibodycontrol, and all experiments were repeated at least 2 times.

Positive binding in CSF and brain extracts was confirmed using antibody6E10.

SPR Analysis of Brain Extracts

4 brain extracts from AD patients and 4 brain extracts from age-matchedcontrols were pooled and analyzed. Brain samples, homogenized in TBS,included frontal cortex Brodmann area 9. All experiments were performedusing a Molecular Affinity Screening System (MASS-1) (Sierra SensorsGmbH, Hamburg, Germany), an analytical biosensor that employs highintensity laser light and high speed optical scanning to monitor bindinginteractions in real time as described in Example 6. Purified antibodiesgenerated for cyclopeptides described herein were captured on separateflow cells of a protein A-derivatized sensor chip and diluted samplesinjected over the surfaces for 180 seconds, followed by 120 seconds ofdissociation in buffer and surface regeneration. Binding responses weredouble-referenced by subtraction of mouse control IgG reference surfacebinding and assay buffer, and the different groups of samples compared.

SPR Analysis of Synthetic Oligomer Binding

Serial 2-fold dilutions (7.8 nM to 2000 nM) of commercially-preparedsynthetic amyloid beta oligomers (SynAging SAS, Vandoeurve-iés-Nancy,France, were tested for binding to covalently immobilized antibodies bySPR. Antibody mAb6E10 and mouse control IgG were used as positive andnegative controls respectively. Monomer binding was assessed in aseparate assay as described in Example 2.

Results CSF, Brain Extracts and Immunohistochemistry

Antibodies were tested for their ability to bind A-beta in CSF, solublebrain extracts and tissue samples of cavaderic AD brains. Strength ofrelative positivity in the tables is shown by the number plus signs.

IHC results are also summarized in Table 1, 3 and 5 where “+/−” denotesstaining similar to or distinct from isotype control but without clearplaque morphology.

FIG. 2A-FIG. 2D shows an example of the lack of plaque staining on freshfrozen sections with antibody 303-25, 301-17 and 305-62 compared to thepositive plaque staining seen with 6E10 antibody.

As shown in Tables 1-6 and FIG. 2A-FIG. 2D, antibodies raised to thecyclopeptides bound to A-beta in brain extracts and/or CSF, but did notappreciably bind to monomers on SPR, and did not appreciably bind toplaque fibrils by IHC. They did bind commercially prepared oligomers ofA-beta preferentially.

Each of the antibodies tested (301-17, 301-3, 301-11, 303-25, 303-26,303-30 305-59, 305-61. 305-62) as well as the positive control antibodybut not the control IgG antibody bound the commercial preparation ofoligomeric A-beta.

TABLE 1 Summary of binding characteristics Oligomers/ CSF Brain ExtractIHC - Plaque Clone # Monomers AD/Non-AD AD/Non-AD Stainingcyclo(CGHHQKG) 301-03 ++ + ++ +/− (SEQ ID NO: 2) 301-11 ++ ++ ++ +/−301-17 ++ + ++ − * Scoring is relative to other clones in the samesample category.

TABLE 2 A-beta Oligomer and Monomer binding Clone tested 301-1D6 (03)301-11 301-17 oligomer 15.07, 13.78* RU 19.27RU   24.01, 34.08 RU*monomer −0.6RU** −0.1 RU** 0.3RU** *the results of two assays usingsynthetic commercially prepared oligomers **monomer preparations weremade and assayed as described in Example 2

Formalin fixed brain tissue of a confirmed AD patient was also tested.The results are shown in Table 7. The brain tissues were fixed in 10%buffered formalin for several days and paraffin processed in the SakuraVIP tissue processors. Tissue sections were probed with 1 μg/ml ofantibody with and without microwave antigen retrieval (AR). Thepan-amyloid beta reactive antibody 6E10 was included along with selectedantibody clones as a positive control. Antibodies were diluted inAntibody Diluent (Ventana), color was developed with OptiView DAB(Ventana). The staining was performed on the Ventana Benchmark XT IHCstainer. Images were obtained with an Olympus BX45 microscope. Imageswere analyzed blind by a professional pathologist with expertise inneuropathology.

TABLE 3 Summary of binding characteristics Oligomers/ CSF Brain ExtractIHC - Plaque Clone # Monomers AD/Non-AD AD/Non-AD Stainingcyclo(CGQKLVG) 305-59 (5G1)  ++ − ++ +/− (SEQ ID NO: 6) 305-61 (7E9)  ++ ++ − − 305-62 (8H10) ++ − + − * Scoring is relative to other clonesin the same sample category

TABLE 4 A-beta Oligomer and Monomer binding Clone tested 305-59 305-61305-62 oligomer 11.73 RU    12.23, 17.53* 8.76, 12.34 RU* monomer −0.2RU** 0.2RU** −0.1** *the results of two assays using syntheticcommercially prepared oligomers **monomer preparations were made andassayed as described in Example 2

TABLE 5 Binding properties Oligomers/ CSF Brain Extract IHC - PlaqueClone # Monomers AD/Non-AD AD/Non-AD Staining cyclo(CGHDSGG) 303-25 +++++ + − (SEQ ID NO: 10) 303-26 + − ++ − 303-30 ++ − ++ +/−

TABLE 6 A-beta Oligomer binding RU values subtracted for monomer bindingClone tested 303-26 303-25 303-30 oligomer 2.97 18.29, 9.43** 13.64Monomer −1.0* −0.2* −0.2* *the results of two assays using syntheticcommercially prepared oligomers **monomer preparations were made andassayed as described in Example 2

TABLE 7 Plaque staining in formalin fixed brain tissues EpitopeAntibodies to test Staining of senile plaque amyloid 301 11 Neg 17 Neg303 25 Neg 305 59 (5G1) possible weak staining 61 (7E9)  Neg  62 (8H10)Neg Positive Control 6E10 strongly positive

Example 7 Inhibition of Oligomer Propagation

The biological functionality of antibodies was tested in vitro byexamining their effects on Amyloid Beta (An) aggregation using theThioflavin T (ThT) binding assay. Aβ aggregation is induced by andpropagated through nuclei of preformed small Aβ oligomers, and thecomplete process from monomeric Aβ to soluble oligomers to insolublefibrils is accompanied by concomitantly increasing beta sheet formation.This can be monitored by ThT, a benzothiazole salt, whose excitation andemission maxima shifts from 385 to 450 nm and from 445 to 482 nmrespectively when bound to beta sheet-rich structures and resulting inincreased fluorescence. Briefly, Aβ 1-42 (Bachem Americas Inc.,Torrance, Calif.) was solubilized, sonicated, diluted in Tris-EDTAbuffer (pH7.4) and added to wells of a black 96-well microtitre plate(Greiner Bio-One, Monroe, N.C.) to which equal volumes of cyclopeptideraised antibody or irrelevant mouse IgG antibody isotype controls wereadded, resulting in a 1:5 molar ratio of Aβ1-42 peptide to antibody. ThTwas added and plates incubated at room temperature for 24 hours, withThT fluorescence measurements (excitation at 440 nm, emission at 486 nm)recorded every hour using a Wallac Victor3v 1420 Multilabel Counter(PerkinElmer, Waltham, Mass.). Fluorescent readings from backgroundbuffer were subtracted from all wells, and readings from antibody onlywells were further subtracted from the corresponding wells.

As shown in FIG. 3A-FIG. 3F, Aβ42 aggregation, as monitored by ThTfluorescence, demonstrated a sigmoidal shape characterized by an initiallag phase with minimal fluorescence, an exponential phase with a rapidincrease in fluorescence and finally a plateau phase during which the Aβmolecular species are at equilibrium and during which there is noincrease in fluorescence. Co-incubation of Aβ42 with an irrelevant mouseantibody did not have any significant effect on the aggregation process.In contrast, co-incubation of Aβ42 with the test antibodies completelyinhibited all phases of the aggregation process. In contrast,co-incubation of Aβ42 with the test antibodies completely inhibited allphases of the aggregation process.

Results obtained with antibody 301-17 (IgG3 isotype) are shown in FIG.3A. Results obtained with antibodies 305-61, 305-62 and 305-64 are shownin FIG. 3B, FIG. 3C and FIG. 3D. 305-59 was also tested and showedsimilar results (FIG. 3F).

Results were also obtained with antibody 303-25 which is shown in FIG.3E. 303-30 was also tested and showed similar results.

As the ThT aggregation assay mimics the in vivo biophysical/biochemicalstages of Aβ propagation and aggregation from monomers, oligomers,protofibrils and fibrils that is pivotal in AD pathogenesis, theantibodies raised to cyclo(CGHHQKG) (SEQ ID NO: 2), cyclo CGQKLVG (SEQID NO: 6) or cyclo(CGHDSGG) (SEQ ID NO: 10), demonstrate the potentialto completely abrogate this process. Isotype control performed usingmouse IgG control showed no inhibition.

Example 8 Toxicity Inhibition Assay

The inhibition of toxicity of A-beta42 oligomers by antibodies raised tothe cyclopeptides were tested in a rat primary cortical neuron assay.

Antibody and control IgG are each adjusted to a concentration such as 2mg/mL. Various molar ratios of A-beta oligomer and antibody are testedalong with a vehicle control, A-beta oligomer alone and a positivecontrol such as the neuroprotective peptide humanin HNG.

An exemplary set up is shown in Table 8.

Following preincubation for 10 minutes at room temperature, the volumeis adjusted to 840 microlitres with culture medium. The solution isincubated for 5 min at 37 C. The solution is then added directly to theprimary cortical neurons and cells are incubated for 24 h. Cellviability can be determined using the MTT assay.

TABLE 8 AβO/AB AβO AβO AB AB Medium Final volume molar ratio (μL) (μM)(μM) (μL) (μL) (μL) 5/1 1.68 4.2 0.84 12.73 185.6 200 1/1 1.68 4.2 4.2063.64 134.7 200 1/2 1.68 4.2 8.4 127.27 71.1 200 AβO working solution:2.2 mg/mL-500 μM CTRL vehicle: 1.68 μL of oligomer buffer + 127.3 μLPBS + 711 μL culture medium CTRL AβO: 1.68 μL of AβO + 127.3 μL PBS +711 μL culture medium CTRL HNG: 1.68 μL of AβO + 8.4 μL HNG (100 nMfinal) + 127.3 μL PBS + 702.6 μL culture medium

In a first assay, the antibody 301-17 alone showed some toxicity at thehighest concentration (1/2 oligomer/antibody ratio), likely due toendotoxin contamination of the antibody preparation, but demonstratedinhibition of A-beta oligomer toxicity when added at lowerconcentrations (1/1 and 5/1 oligomer/antibody ratios) (FIG. 4B).

The assay was repeated using an antibody preparation generated under lowendotoxin conditions. This time, in the absence of A-beta oligomers, theantibody alone had no effect on neuronal cell viability. When incubatedin the presence of A-beta oligomers, the antibody inhibited A-betaoligomer-induced neuronal death at all molar ratios tested (FIG. 5A).The assay was repeated using recombinant 301-17 antibody grafted to amouse IgG1 backbone. Again, in the absence of A-beta oligomers, theantibody alone had no effect on neuronal cell viability. When incubatedin the presence of A-beta oligomers, the antibody inhibited A-betaoligomer-induced neuronal death at all molar ratios tested (FIG. 5B).

Antibody 301-3 was also tested in the assay. Like 301-17, antibody aloneshowed no toxicity to neuronal cell viability whereas when antibody whenincubated in the presence of A-beta oligomers, the antibody inhibitedA-beta oligomer-induced neuronal death at all molar ratios tested (FIG.4E).

This test was also conducted using antibody 305-62. The antibody aloneshowed no toxicity (FIG. 4C) and inhibition of A-beta oligomer toxicitywas observed for all concentrations of antibody to oligomer: 1:5, 1:1and 2:1 (FIG. 4C). This test was also conducted using antibody 305-61.

The antibody alone showed no toxicity (FIG. 4D) and inhibition of A-betaoligomer toxicity was observed for all concentrations of antibody tooligomer: 1:5, 1:1 and 2:1 (FIG. 4D). The presence of antibody 301-61resulted in a dose-dependent protection toward AβO-induced cell death(FIG. 4D). The maximal neuroprotection was reached at a molar ratio of1:2 (AβO:AB1) resulting in a remaining cell viability of 80.1±3.8% ofcontrol.

The test was also conducted using antibody 303-25. The antibody aloneshowed no toxicity (FIG. 4F) and inhibition of A-beta oligomer toxicitywas observed for all concentrations of antibody to oligomer: 1:5, 1:1and 2:1 (FIG. 4F). In the absence of AβO, antibody 303-25 had no effecton cell viability (FIG. 4F). The presence of antibody 3 resulted in adose-dependent protection toward AβO-induced cell death. The maximalneuroprotection was reached at a molar ratio of 1:2 (AβO:AB1) resultingin a remaining cell viability of 88.8±4.8% of control.

Controls are shown in FIG. 4A. (NB: FIG. 4B, FIG. 4C and FIG. 4E showdecreasing ratio of test antibody:oligomer whereas FIG. 4D and FIG. 4Fshow an increasing ratio of test antibody:oligomer).

Example 9

RT-PCR was carried out using 5′ RACE and gene specific reverse primerswhich amplify the appropriate mouse immunoglobulin heavy chain(IgG1/IgG3/IgG2A) and light chain (kappa) variable region sequences.

The specific bands were excised and cloned into pCR-Blunt II-TOPO vectorfor sequencing, and the constructs were transformed into E. coli

At least 8 colonies of each chain were picked & PCR screened for thepresence of amplified regions prior to sequencing. Selected PCR positiveclones were sequenced. The complementarity determining regions (CDRs)are identified according to IMGT/LIGM-DB.

CDR Sequencing—Cyclo(CGHHQKG) Antibodies (SEQ ID NO: 2)

The CDR sequences of 301-17 which was determined to have an IgG3 heavychain and a kappa light chain are in Table 9A. The consensus DNAsequence and protein sequences of the variable portion of the heavy andlight chain are provided in Table 10A.

The CDR sequences of 301-11 are in Table 9B. The consensus DNA sequenceand protein sequences of the variable portion of the heavy and lightchain are provided in Table 10A.

The CDR sequences of antibody 301-03 (1 and 2) raised againstcyclo(CGHHQKG) are provided in Table 9C. The consensus DNA sequence andprotein sequences of the variable portion of the heavy and light chainare provided in Table 10A.

CDR Sequencing—Cyclo(CGQKLVG) Antibodies (SEQ ID NO: 6)

The CDR sequences of 305-61 (7E9.1) which was determined to have an IgG3heavy chain and a kappa light chain are in Table 9D. The consensus DNAsequence and protein sequences of the variable portion of the heavy andlight chain are provided in Table 10B.

The CDR sequences of 305-62 (8H10) which was determined to have an IgG1heavy chain and a kappa light chain are in Table 9E. The consensus DNAsequence and protein sequences of the variable portion of the heavy andlight chain are provided in Table 10B.

The CDR sequences of 305-59 which was determined to have an IgG1 heavychain and a kappa light chain are in Table 9I. The consensus DNAsequence and protein sequences of the variable portion of the heavy andlight chain are provided in Table 10B.

CDR Sequencing—Cyclo(CGHDSGG) Antibodies (SEQ ID NO: 10)

The CDR sequences of 303-25 which was determined to have an IgG1 heavychain and a kappa light chain are in Table 9F. The consensus DNAsequence and protein sequences of the variable portion of the heavy andlight chain are provided in Table 10C.

The CDR sequences of 303-26 (3 kappa chains) and 303-30 are in Table 9Gand 9H respectively. The consensus DNA sequence and protein sequences ofthe variable portion of the heavy and light chain are provided in Table10C.

Table 9

TABLE 9A CDR sequences of antibody 301-17 raised against cyclo (CGHHQKG)Chain CDR Sequence SEQ ID NO. Heavy CDR-H1 GYSFTSYW 20 CDR-H2 VHPGRGVST21 CDR-H3 SRSHGNTYWFFDV 22 Light CDR-L1 QSIVHSNGNTY 23 CDR-L2 KVS 24CDR-L3 FQGSHVPFT 25

TABLE 9B CDR sequences of antibody 301-11 raised against cyclo (CGHHQKG)Chain CDR Sequence SEQ ID NO. Heavy CDR-H1 GFTFSDYY 26 CDR-H2 ISDGGSYT27 CDR-H3 ARDYYGSSSYTSGFAY 28 Light CDR-L1 QSLLNSRTRKNY 29 CDR-L2 WAS 30CDR-L3 KQSYNLYT 31

TABLE 9C CDR sequences of antibody 301-03 (1 and 2) raised againstcyclo(CGHHQKG) Chain CDR Sequence SEQ ID NO. Heavy CDR-H1 GFTFSDYY 32CDR-H2 ISDGGSYT 33 CDR-H3 ARDYYGSNSYTSGFAY 34 Light CDR-L1 QSLLNSRTRKNY35 CDR-L2 WAS 36 CDR-L3 KQSYNLYT 37 Light CDR-L1 QSIVHSNGNTY 38 CDR-L2KVS 39 CDR-L3 FQGSHVPLT 40

TABLE 9D CDR sequences of antibody 305-61 (7E9) raised againstcyclo(CGQKLVG) Chain CDR Sequence SEQ ID NO. Heavy CDR-H1 GYTFTDYE 41CDR-H2 IDPETGDT 42 CDR-H3 TSPIYYDYDWFAY 43 Light CDR-L1 QSLLNNRTRKNY 44CDR-L2 WAS 5 CDR-L3 KQSYNLRT 46

TABLE 9E CDR sequences of antibody 305-62 (8H10) raised againstcyclo(CGQKLVG) Chain CDR Sequence SEQ ID NO Heavy CDR-H1 GFSLSTSGMG 47CDR-H2 IWWDDDK 48 CDR-H3 ARSITTVVATPFDY 49 Light CDR-L1 QNVRSA 50 CDR-L2LAS 51 CDR-L3 LQHWNSPFT 52

TABLE 9F CDR sequences of antibody 303-25 raised against cyclo(CGHDSGG)Chain CDR Sequence SEQ ID NO Heavy CDR-H1 GYTFTSYW 53 CDR-H2 IDPSDSQT 54CDR-H3 SRGGY 55 Light CDR-L1 QDINNY 56 CDR-L2 YTS 57 CDR-L3 LQYDNLWT 58

TABLE 9G CDR sequences of antibody 303-26 raised against cyclo(CGHDSGG)Chain CDR Sequence SEQ ID NO Heavy CDR-H1 GYTFTSYW 59 CDR-H2 IDPSDSET 60CDR-H3 TRGTY 61 Light 1 CDR-L1 QSVSTSSYSY 62 CDR-L2 YAS 63 CDR-L3QHSLEIPWT 64 Light 2 CDR-L1 SSVSSAY 65 CDR-L2 STS 66 CDR-L3 HQYHRSPFT 67Light 3 CDR-L1 SQDINKYIAWY 68 CDR-L2 NTS 69 CDR-L3 LQHDNLWT 70

TABLE 9H CDR sequences of antibody 303-30 raised against cyclo(CGHDSGG)Chain CDR Sequence SEQ ID NO Heavy CDR-H1 GYTFTSYW 71 CDR-H2 IDPSDSET 72CDR-H3 TRGTY 73 Light 1 CDR-L1 QSVSTSSYSY 74 CDR-L2 YAS 75 CDR-L3QHSLEIPWT 76 Light 2 CDR-L1 SSVSSAY 77 CDR-L2 STS 78 CDR-L3 HQYHRSPFT 79Light 3 CDR-L1 SQDINKYIAWY 80 CDR-L2 NTS 81 CDR-L3 LQHDNLWT 82

TABLE 9I CDR sequences of antibody 305-59 raised against cyclo(CGQKLVG)Chain CDR Sequence SEQ ID NO Heavy CDR-H1 GFNIKDTY 185 CDR-H2 IAPASGNT186 CDR-H3 ARHVY 187 Light CDR-L1 QSVSND 188 CDR-L2 YAS 189 CDR-L3QQDYISPYT 190

Table 10

TABLE 10A Heavy chain and light chain variable sequences of antibodiesraised against cyclo(CGHHQKG) Consensus DNA sequence and translatedprotein sequences of the variable region. The complementaritydetermining regions (CDRs) are underlined according to IMGT/LIGM-DB.Isotype Consensus DNA Sequence Protein sequence 301-17ATGGGATGGAGCTGTATCATCCTCTTTTTGGTAGCAACAGCTACAGGTGTCCACTCMGWSCIILFLVATATGVHSQ IgG3CCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAVQLQQPGAELVKPGASVKMS SEQ ID NO: 83, AAATGTCCTGCAAGGCTTCTGGCTACAGCTTCACCAGCTACTGG ATAAACTGGGTG CKAS GYSFTSYW INWVKQRP 84AAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAT GTTCATCCTGGTAGAGG GQGLEWIGDVHPGRGVST YN TGTTTCTACC TACAATGCGAAGTTCAAGAGCAAGGCCACACTGACTCTAGACACGTAKFKSKATLTLDTSSSTAYMCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTC QLSSLTSEDSAVYYCSRSHG TATTATTGT TCAAGATCCCACGGTAATACCTACTGGTTCTTCGATGTC TGGGGCGCNTYWFFDV WGAGTTVTVSSA AGGGACCACGGTCACCGTCTCCTCAGCTACAACAACAGCCCCATCTTTTAPS 301-17 ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGMKLPVRLLVLMFWIPASSSD KappaTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGVLMTQTPLSLPVSLGDQASI SEQ ID NO: 85, CCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTAT SCRSS QSIVHSNGNTY LEWY 86TTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAA AGT LQKPGQSPKLLIYKVS NRFS TTCC AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGGVPDRFSGSGSGTDFTLKISATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGC RVEAEDLGVYYCFQGSHVPF TTTCAAGGTTCACATGTTCCATTCACG TTCGGCTCGGGGACAAAGTTGGAAATAAA TFGSGTKLEIKRADA ACGGGCTGATGCT 301-11ATGAACTTTGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGMNFGLSLIFLVLVLKGVQCE IgG3TGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAVQLVESGGGLVKPGGSLKLS SEQ ID NO: 87, AACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTAC ATGTATTGGGTT CAAS GFTFSDYY MYWVRQTP 88CGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACC ATTAGTGATGGTGGTAG EKRLEWVATISDGGSYT SYP TTACACC TCCTATCCAGACAGTGTGAAGGGACGATTCACCATCTCCAGAGACAATGDSVKGRFTISRDNAKNNLYLCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACAGCCATG QMSSLRSEDTAMYYCARDYY TATTACTGT GCAAGAGATTACTACGGTAGTAGTAGCTACACCTCGGGCTTTGCTTAGSSSYTSGFAY WGQGTLVTV C TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA SA 301-11ATGGATTCACAGGCCCAGGTTCTTATATTGCTGCTGCTATGGGTATCTGGTACCTGMDSQAQVLILLLLWVSGTCG KappaTGGGGACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAACAGGAGAGADIVMSQSPSSLAVSTGEKVT SEQ ID NO: 89, AGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAG MSCKSS QSLLNSRTRKNY LA 90 AACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTA WYQQKPGQSPKLLIY WASTR C TGGGCATCC ACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGESGVPDRFTGSGSGTDFTLTGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTAT ISSVQAEDLAVYYCKQSYNL TACTGC AAGCAATCTTATAATCTGTACACG TTCGGAGGGGGGACCAAGCTGGAAAT YTFGGGTKLEIK AAAA 301-03ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGMNFGLSLIFLVLVLKGVQCE IgG3TGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAVQLVESGGGLVKPGGSLKLS SEQ ID NO: 91, AACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTAC ATGTATTGGGTT CAAS GFTFSDYY MYWVRQTP 92CGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACC ATTAGTGATGGTGGTAG EKRLEWVATISDGGSYT SYP TTACACC TCCTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAGTGDSVKGRFTISRDSAKNNLYLCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATG QMSSLKSEDTAMYYCARDYY TATTACTGT GCAAGAGATTACTACGGTAGTAATAGTTACACCTCGGGCTTTGCTTAGSNSYTSGFAY WGQGTLVTV C TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA SA 301-03ATGGATTCACAGGCCCAGGTTCTTATATTGCTGCTGCTATGGGTATCTGGTACCTGMDSQAQVLILLLLWVSGTCG Kappa 1TGGGGACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGADIVMSQSPSSLAVSAGEKVT SEQ ID NO: 93, AGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAATAGTAGAACCCGAAAG MSCKSS QSLLNSRTRKNY LA 94 AACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTA WYQQKPGQSPKLLIY WASTR C TGGGCATCC ACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGESGVPDRFTGSGSGTDFTLTGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTAT ISSVQAEDLAVYYCKQSYNL TACTGC AAGCAATCTTATAATCTGTACACG TTCGGAGGGGGGACCAAGCTGGAAAT YTFGGGTKLEIK AAAA 301-03ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGMKLPVRLLVLMFWIPASSSD Kappa 2TGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGVLMTQTPLSLPVSLGDQASI SEQ ID NO: 95, CCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTAT SCRSS QSIVHSNGNTY LEWY 96TTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTAC AAAGT LQKPGQSPKLLIYKVS NRFS TTCC AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGGVPDRFSGSGSGTDFTLKISATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGC RVEAEDLGVYFCFQGSHVPL TTTCAAGGTTCACATGTTCCTCTCACG TTCGGTGCTGGGACCAAGCTGGAGCTGAA TFGAGTKLELK A

TABLE 10B Heavy chain and light chain variable sequences of an antibodyraised against cyclo(CGQKLVG) Consensus DNA sequence and translatedprotein sequences of the variable region. The complementaritydetermining regions (CDRs) are underlined according to IMGT/LIGM-DB.Isotype Consensus DNA Sequence Protein sequence 305-61ATGGAATGGAGCTGGGTCTTTCTCTTCCTCCTGTCAGTAATTGCA MEWSWVFLFLLSVIAG IgG3GGTGTCCAATCCCAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTG VQSQVQLQQSGAELVR SEQ IDNO: GTGAGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCG GGC PGASVTLSCKAS GYTF 97,98 TACACATTTACTGACTAT GAAATGCACTGGGTGAAGCAGACACCT TDYE MHWVKQTPVHGLGTGCATGGCCTGGAATGGATTGGAGCT ATTGATCCTGAAACTGGT EWIGA IDPETGDT AYN GATACTGCCTACAATCAGGAGTTCAAGGGCAAGGCCACACTGACT QEFKGKATLTADKSSSGCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTG TAYMELRSLTSEDSAVACATCTGAGGACTCTGCCGTCTATTACTGT ACAAGCCCCATCTAC YYC TSPIYYDYDWFAYTATGATTACGACTGGTTTGCTTAC TGGGGCCACGGGACTCTGGTC WGHGTLVTVSAATTTAACTGTCTCTGCAGCTACAACAACAGCCCCATCT PS 305-61ATGGATTCACAGGCCCAGGTTCTTATATTGCTGCTGCTATGGGTA MDSQAQVLILLLLWVS KappaTCTGGTACCTGTGGGGACATTGTGATGTCACAGTCTCCATCCTCC GTCGDIVMSQSPSSLA SEQ IDNO: CTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCC VSAGEKVTMSCKSS QS 99,100 AGT CAGAGTCTGCTCAACAATAGAACCCGAAAGAACTAC TTGGCT LLNNRTRKNY LAWYQQTGGTAC CAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTAC KPGQSPKLLIY WAS TRTGGGCATCC ACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGC ESGVPDRFTGSGSGTDAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAG FTLTISSVQAEDLAVYGCTGAAGACCTGGCAGTTTATTACTGC AAGCAATCTTATAATCTT YC KQSYNLRT FGGGTK CGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGAT LEIKRADA GCT 305-62ATGGACAGGCTTACTTCTTCATTCCTGCTGCTGATTGTCCCTGCA MDRLTSSFLLLIVPAY IgG1TATGTCTTGTCCCAAGTTACTCTAAAAGAGTCTGGCCCTGGGATA VLSQVTLKESGPGILK SEQ IDNO: TTGAAGCCCTCACAGACCCTCAGTCTGACTTGTTCTTTCTCT GGG PSQTLSLTCSFS GFSL101, 102 TTTTCACTGAGCACTTCTGGTATGGGT GTAGGCTGGATTCGTCAG STSGMGVGWIRQPSGK CCTTCAGGGAAGGGTCTGGAGTGGCTGGCACAC ATTTGGTGGGAT GLEWLAHIWWDDDK YY GATGATAAG TACTATAACCCATCCCTGAAGAGCCAGCTCACAATCNPSLKSQLTISKDTSR TCCAAGGATACCTCCAGAAACCAGGTATTCCTCAAGATCACCAGTNQVFLKITSVDTADTA GTGGACACTGCAGATACTGCCACTTACTACTGT GCTCGAAGTATT TYYCARSITTVVATPF ACTACGGTAGTAGCTACGCCCTTTGACTAC TGGGGCCAAGGCACC DYWGQGTTLTVSSAKT ACTCTCACAGTCTCCTCAGCCAAAACGACAC T 305-62ATGGGCATCAAGATGGAGTTTCAGACCCAGGTCTTTGTATTCGTG MGIKMEFQTQVFVFVL KappaTTGCTCTGGTTGTCTGGTGTTGATGGAGACATTGTGATGACCCAG LWLSGVDGDIVMTQSQ SEQ IDNO: TCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATC KFMSTSVGDRVSITCK 103,104 ACCTGCAAGGCCAGT CAGAATGTTCGTTCTGCT GTAGCCTGGTAT AS QNVRSA VAWYQQKPCAACAGAAACCAGGGCAGTCTCCTAAAGCACTGATTTAC CTGGCA GQSPKALIY LAS NRHT TCCAACCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGA GVPDRFTGSGSGTDFTTCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCATTCTGAA LTISNVHSEDLTDYFCGACCTGACAGATTATTTCTGT CTGCAACATTGGAATTCTCCGTTC LQHWNSPFT FGGGTKL ACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCT EIKRADA 305-59ATGAAATTCAGCTGGGTCATCTTCTTCCTGATGGCAGTGGTTACA MKFSWVIFFLMAVVTG IgG1GGGGTCAATTCAGAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTT VNSEVQLQQSGAELVK SEQ IDNO: GTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGTTTCT GGC PGASVKLSCTVS GFNI191, 192 TTCAACATTAAAGACACCTAT GTGCACTGGGTGAAGCAGAGGC KDTY VHWVKQRPEQGLCTGAACAGGGCCTGGAGTGGATTGGAAGG ATTGCTCCTGCGAG EWIGR IAPASGNT KYTGGTAATACT AAATATGCCCCGAATTTCCAGGACAAGGCCACTA APNFQDKATITADTSSTAACAGCGGACACATCCT CCAACACAGCCTACCTGCAGCTCAACA NTAYLQLNSLTSEDTAGCCTGACATCTGAGGACACTGCCGTCTATTACTGT GCGCGTCAC VYYC ARHVY WGQGTLV GTCTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA TVSA 305-59ATGAAGTCACAGACCCAGGTCTTCGTATTTCTACTGCTCTGTGTG MKSQTQVFVFLLLCVS kappaTCTGGTGCTCATGGGAGTATTGTGATGACCCAGACTCCCAAATTC GAHGSIVMTQTPKFLL SEQ IDCTGCTTGTATCAGCAGGAGACAGGGTTACCATAACCTGCAAGGCC VSAGDRVTITCKAS QS NO 193,194 AGT CAGAGTGTGAGTAATGAT GTAGTTTGGTACCAACAGAAGC VSND VVWYQQKPGQSPCAGGGCAGTCTCCTAAACTGCTGATATAC TATGCATCC AATCGC KLLIY YAS NRYTGVPDTACACTGGAGTCCCTGATCGCTTTACTGGCAGTGGATATGGGACG RFTGSGYGTDFTFTISGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAAGACCTGGCA TVQAEDLAVYFC QQDYGTTTATTTCTGT CAGCAGGATTATATCTCTCCGTACACG TTC ISPYT FGGGTKLEIKGGAGGGGGGACCAAGCTGGAAATAAAA

TABLE 10C Heavy chain and light chain variable sequences of an antibodyraised against cyclo(CGHDSGG) Consensus DNA sequence and translatedprotein sequences of the variable region. The complementaritydetermining regions (CDRs) are underlined according to IMGT/LIGM-DB.Isotype Consensus DNA Sequence Protein sequence 303-25ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACA MGWSCIILFLVATATG IgG2aGGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTG VHSQVQLQQPGAELVR SEQ IDNO: GTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCT GGC PGASVKLSCKAS GYTF105, 106 TACACCTTCACCAGCTACTGG ATGAACTGGGTGAAGCAGAGGCCT TSYWMNWVKQRPGQGL GGACAAGGCCTTGAATGGATTGGTATG ATTGATCCTTCAGACAGT EWIGMIDPSDSQT HYN CAAACT CACTACAATCAAATGTTCAAGGACAAGGCCACATTGACTQMFKDKATLTVDKSSS GTAGACAAATCCTCCAGCACAGCCTACCTGCAGCTCAGCAGCCTGTAYLQLSSLTSEDSAV ACATCTGAGGACTCTGCGGTCTATTACTGTTCAAGAGGGGGCTAC YYCSRGGYW GQGTTLT TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA VSS KappaATGAGACCGTCTATTCAGTTCCTGGGGCTCTTGTTGTTCTGGCTT MRPSIQFLGLLLFWLH SEQ IDNO: CATGGTGCTCAGTGTGACATCCAGATGACACAGTCTCCATCCTCA GAQCDIQMTQSPSSLS 107,108 CTGTCTGCATCTCTGGGAGGCAAAGTCACCATCACTTGCAAGGCA ASLGGKVTITCKAS QD AGCCAAGACATTAACAACTATAT AGCTTGGTACCAACACAAGCCT INNY IAWYQHKPGKGPGGAAAAGGTCCTAGGCAGCTCATATAT TACACATCT ACATTGCAG RQLIY YTS TLQPGIPSCCAGGCATCCCATCAAGGTTCAGTGGAAGTGGGTCTGGGAGAGAT RFSGSGSGRDYSFTISTATTCCTTCACCATCAGCGACCTGGAGCCTGAAGATATTGCAACT DLEPEDIATYYC LQYDTATTATTGT CTACAGTATGATAATCTGTGGACG TTCGGTGGAGGC NLWT FGGGTKLEIKACCAAGCTGGAAATCAAA 303-26 ATGGGATGGAGCTGTATCATCCTCTTTTTGGTAGCAACAGCTACAMGWSCIILFLVATATG IgG1 GGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGVHSQVQLQQPGAELVR SEQ ID NO: GTGAGGCCTGGGTCTTCAGTGAAGCTGTCCTGCAAGGCTTCTggc PGSSVKLSCKAS GYTF 109, 110 tacaccttcaccagctactggATGAACTGGGTGAAGCAGAGGCCT TSYW MNWVKQRPGQGL GGACAAGGCCTTGAATGGATTGGTATGattgatccttcagacagt EWIGM IDPSDSET HYN gaaactCACTACAATCAAATGTTCAAGGACAAGGCCACATTGACT QMFKDKATLTVDKSSSGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTG TAYMQLSSLTSEDSAVACATCTGAGGACTCTGCGGTCTATTACTGT acaagagggacttac YYC TRGTY WGQGTQVTTGGGGCCAAGGGACTCAGGTCACTGTCTCTGCA VSA Kappa 1ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTT METDTLLLWVLLLWVP SEQ IDNO: CCAGGTTCCACTGGTGACATTGTGCTGACACAGTCTCCTGCTTCC GSTGDIVLTQSPASLA 111,112 TTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCC VSLGQRATISCRAS QS AGCcaaagtgtcagtacatctagctatagttat ATGCACTGGTAC VSTSSYSY MHWYQQKPCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCAAG tatgca GQPPKLLIK YAS NLES tccAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGG GVPARFSGSGSGTDFTTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAG LNIHPVEEEDTATYYCGATACTGCAACATATTACTGT cagcacagtttggagattccgtgg QHSLEIPWT FGGGTKL acgTTCGGTGGAGGCACCAAGCTGGAAATCAAA EIK Kappa 2ATGGATTTTCAGGTGCAGATTTTCAGCTTCATGCTAATCAGTGCC MDFQVQIFSFMLISAS SEQ IDNO: TCAGTCATAATGTCCAGAGGACAAATTGTTCTCACCCAGTCTCCA VIMSRGQIVLTQSPAI 113,114 GCAATCATGTCTGCATCTCTAGGGGAACGGGTCACCATGACCTGC MSASLGERVTMTCTASACTGCCAGC tcaagtgttagttccgcttac TTGCACTGGTACCAG SSVSSAY LHWYQQKPGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTAT agcacatcc SSPKLWIY STS NLASGAACCTGGCTTCTGGAGTCCCAACTCGCTTCAGTGGCAGTGGATCT VPTRFSGSGSGTSYSLGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGAT TISSMEAEDAATYYC HGCTGCCACTTATTACTGC caccagtatcatcgttccccgttcacg QYHRSPFT FGAGTKLETTCGGTGCTGGGACCAAGCTGGAGCTGAAA LK Kappa 3ATGAGACCGTCTATTCAGTTCCTGGGGCTCTTGTTGTTCTGGCTT MRPSIQFLGLLLFWLH SEQ IDNO: CATGGTGCTCAGTGTGACATCCAGATGACACAGTCTCCATACTCA GAQCDIQMTQSPYSLS 115,116 CTGTCTGCATCTCTGGGAGGCAAAGTCACCATCACTTGCAAGGCA ASLGGKVTITCKA SQDagccaagacattaacaagtatatagcttggtac CAACACAAGCCT INKYIAWY QHKPGKGPGGAAAAGGTCCTAGGCTGCTCATACAT aacacatct ACATTACAG RLLIH NTS TLQPGIPSCCAGGCATCCCATCAAGGTTCAGTGGAAGTGGGTCTGGGAGAGAT RFSGSGSGRDYSFSISTATTCCTTCAGCATCAGCAACCTGGAGCCTGAAGATATTGCAACT NLEPEDIATYYC LQHDTATTATTGT ctacagcatgataatctgtggacg TTCGGTGGAGGC NLWT FGGGTKLEIKACCAAGCTGGAAATCAAA 303-30 ATGGGATGGAGCTGTATCATCCTCTTTTTGGTAGCAACAGCTACAMGWSCIILFLVATATG IgG1 GGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGVHSQVQLQQPGAELVR SEQ ID NO: GTGAGGCCTGGGTCTTCAGTGAAGCTGTCCTGCAAGGCTTCTggc PGSSVKLSCKAS GYTF 117, 118 tacaccttcaccagctactggATGAACTGGGTGAAGCAGAGGCCT TSYW MNWVKQRPGQGL GGACAAGGCCTTGAATGGATTGGTATGattgatccttcagacagt EWIGM IDPSDSET HYN gaaactCACTACAATCAAATGTTCAAGGACAAGGCCACATTGACT QMFKDKATLTVDKSSSGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTG TAYMQLSSLTSEDSAVACATCTGAGGACTCTGCGGTCTATTACTGT acaagagggacttac YYC TRGTY WGQGTQVTTGGGGCCAAGGGACTCAGGTCACTGTCTCTGCA VSA Kappa 1ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTT METDTLLLWVLLLWVP SEQ IDNO: CCAGGTTCCACTGGTGACATTGTGCTGACACAGTCTCCTGCTTCC GSTGDIVLTQSPASLA 119,120 TTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCC VSLGQRATISCRAS QS AGCcaaagtgtcagtacatctagctatagttat ATGCACTGGTAC VSTSSYSY MHWYQQKPCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCAAG tatgca GQPPKLLIK YAS NLES tccAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGG GVPARFSGSGSGTDFTTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAG LNIHPVEEEDTATYYCGATACTGCAACATATTACTGT cagcacagtttggagattccgtgg QHSLEIPWT FGGGTKL acgTTCGGTGGAGGCACCAAGCTGGAAATCAAA EIK Kappa 2ATGGATTTTCAGGTGCAGATTTTCAGCTTCATGCTAATCAGTGCC MDFQVQIFSFMLISAS SEQ IDNO: TCAGTCATAATGTCCAGAGGACAAATTGTTCTCACCCAGTCTCCA VIMSRGQIVLTQSPAI 121,122 GCAATCATGTCTGCATCTCTAGGGGAACGGGTCACCATGACCTGC MSASLGERVTMTCTASACTGCCAGC tcaagtgttagttccgcttac TTGCACTGGTACCAG SSVSSAY LHWYQQKPGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTAT agcacatcc SSPKLWIY STS NLASGAACCTGGCTTCTGGAGTCCCAACTCGCTTCAGTGGCAGTGGATCT VPTRFSGSGSGTSYSLGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGAT TISSMEAEDAATYYC HGCTGCCACTTATTACTGC caccagtatcatcgttccccgttcacg QYHRSPFT FGAGTKLTTTCGGTGCTGGGACCAAGCTGGAGCTGAAALK Kappa 3ATGAGACCGTCTATTCAGTTCCTGGGGCTCTTGTTGTTCTGGCTT MRPSIQFLGLLLFWLH SEQ IDNO: CATGGTGCTCAGTGTGACATCCAGATGACACAGTCTCCATACTCA GAQCDIQMTQSPYSLS 123,124 CTGTCTGCATCTCTGGGAGGCAAAGTCACCATCACTTGCAAGGCA ASLGGKVTITCKA SQDagccaagacattaacaagtatatagcttggtac CAACACAAGCCT INKYIAWY QHKPGKGPGGAAAAGGTCCTAGGCTGCTCATACAT aacacatct ACATTACAG RLLIH NTS TLQPGIPSCCAGGCATCCCATCAAGGTTCAGTGGAAGTGGGTCTGGGAGAGAT RFSGSGSGRDYSFSISTATTCCTTCAGCATCAGCAACCTGGAGCCTGAAGATATTGCAACT NLEPEDIATYYC LQHDTATTATTGT ctacagcatgataatctgtggacg TTCGGTGGAGGC NLWT FGGGTKLEIKACCAAGCTGGAAATCAAA

TABLE 11 A-beta Sequences and compounds comprising linkers 1) (SEQ IDNO: 1) HHQK (SEQ ID NO: 2) CGHHQKG, cyclo(CGHHQKG) (SEQ ID NO: 3)CHHQKG, C-PEG2-HHQKG, cyclo(C-PEG2-HHQKG) (SEQ ID NO: 4) CGHHQK,CGHHQK-PEG2, cyclo(CGHHQK-PEG2) (SEQ ID NO: 5) QKLV (SEQ ID NO: 6)CGQKLVG, cyclo(CGQKLVG) (SEQ ID NO: 7) CQKLVG, C-PEG2-QKLVG,cyclo(C-PEG2-QKLVG) (SEQ ID NO: 8) CGQKLV, CGQKLV-PEG2,cyclo(CGQKLV-PEG2) (SEQ ID NO: 9) HDSG (SEQ ID NO: 10) CGHDSGG,cyclo(CGHDSGG) (SEQ ID NO: 11) CHDSGG, C-PEG2-HDSGG, cyclo(C-PEG2-HDSGG)(SEQ ID NO: 12) CGHDSG, CGHDSG-PEG2, cyclo(CGHDSG-PEG2) 2) (SEQ ID NO:13) HQKLVFFAED (SEQ ID NO: 14) HHQKLVFFAEDVGSNK (SEQ ID NO: 15) HQKLV(SEQ ID NO: 16) HHQKLV (SEQ ID NO: 17) HQKLVF (SEQ ID NO: 18) HQKLVFFHuman A-beta 1-42 (SEQ ID NO: 19)DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

Example 10 Mouse Novel Recognition Assay

The Novel Object Recognition (NOR) model utilizes the normal behavior ofrodents to investigate novel objects for a significantly longer timethan known objects. This test assesses recognition memory for items andits human equivalent is the visual pairwise-comparison (VPC).Recognition of objects is mediated by the perirhinal cortex in rodents,primates and humans. AD pathology develops first in the perirhinal andenthorinal cortex before the hippocampus. The VPC task detects memorydeficit in mild cognitive impairment (MCI) and conversion from MCI to ADis predicted by this task.

The assay was performed by (SynAging SAS, Vandoeurve-iés-Nancy, France).Twelve C57BL6J mice per group (11-12 weeks old) were ICV-injected withvehicle (buffer used for Aβ oligomerization) or AβO (50 pmoles) in thepresence of vehicle (PBS) or antibody (e.g. 301-17, 303-25 or 305-61) onday 0. The cognitive performance of all mice was determined by a NovelObject Recognition (NOR) test performed at days +7 and +8.

The study, done in blind to the operators, involved a total of 48 micedivided in four experimental groups with 12 mice per experimental group.All animals received a single (and unilateral) ICV injection of vehicleOR AβO in the absence or presence of antibody in a total volume of 5 μL.The experimental groups were defined as follow:

GROUP A (vehicle CTRL): ICV injection of vehicle (n=12)

GROUP B (AβO CTRL): ICV injection of AβO (n=12)

GROUP C (Antibody CTRL): ICV injection of AβO+antibody (n=12)

GROUP D (Treatment): ICV injection of AβO+antibody (n=12)

Because a maximum of 24 mice can be ICV injected per day, the injectionswere performed in two independent runs. Six mice from all groups wereincluded in each run.

Before ICV injection, 4 μL of antibody 1 (e.g. 112 pmoles) wereincubated for 30 minutes at room temperature with 1 μL vehicle (i.e.buffer for Aβ oligomerization) or 1 μL AβO (50 pmoles) corresponding toan antibody/AβO molar ratio of 2.24 mouse monoclonal 301-17 antibody.Other ratios as noted were used in other experiments with otherantibodies.

At day 0, mice received a single 5 μL ICV injection of vehicle or AβO inthe presence of vehicle or antibody. The stereotactic coordinates were:from the Bregma (in mm): AP −0.22, L −1.0 and D 2.5. Under anesthesia(ip injection of a mixture of ketamine/xylasine at a dose of 110 and 15mg/kg, respectively), 5 μL were injected into the right lateralventricle. Injections are made using 10 μL Hamilton micro syringesfitted with a 26-gauge needle. The procedure is terminated by asubcutaneous injection of metacam (analgesia) at a dose of 5 mg/kg.Animals are then placed individually in their home cage and the cage isplaced in a heated cabinet until the animal has fully recovered. Animalsare carefully monitored to control recovery after anesthesia.

The NOR test was conducted in one trial with all 48 mice at days +7 and+8. One day before the cognitive test (i.e. at Day +7), mice arehabituated during a 10 min trial during which they are placed in anempty open field. The day of the cognitive test (i.e. Day +8), animalsare placed in the same open field and are allowed to explore freely twoidentical objects for a trial of five minutes (acquisition trial). Thenthe animals are returned to their home cage for an inter-trial time offive minutes. During the retention trial, animals are allowed to exploretwo different objects: the same familiar object and one novel object.During this time, the experimenter, blind to the treatment, records thetime the mouse is actively exploring each object. All trials are videorecorded (Smart v3.0 software, Bioseb). A discrimination index (DI) isthen generated: (DI)=(time exploring novel object−time exploringfamiliar object)/total exploration time. If the total exploration timeis ≤5 s, animals are excluded from the calculation of the discriminationindex and statistical analysis.

Data Analysis:

Graphpad/Prism computer software is used for the statistical analyses. Anon-parametric analysis of variance (Kruskal Wallis test) is carried outfollowed by non-parametric Mann-Whitney U tests in order to comparebetween groups. Values of p<0.05 are considered statisticallysignificant. Data are presented as mean DI±SEM.

Example 11

The novel recognition test described in Example 10 was used to test anantibody raised using cyclo(CGHHQKG) (301-17). As mentioned above,before ICV injection, 4 μL of antibody 1 (112 pmoles) were incubated for30 minutes at room temperature with 1 μL vehicle (i.e. buffer for Aβoligomerization) or 1 μL AβO (50 pmoles) corresponding to anantibody/AβO molar ratio of 2.24.

Results:

Mice from the vehicle control group (Group A) exhibited normal behaviorwith a mean discrimination index of 0.443±0.053 (FIG. 6A). These resultsare in agreement with previous observations of similar control groups atSynAging. As expected, a single ICV injection of AβO (Group B) resultedin a significant impairment (p<0.0001) of the cognitive performance whencompared to vehicle control mice; with a mean discrimination index of−0.062±0.048. AβO-injected mice were not able to discriminate betweennovel and familiar objects (FIG. 6A).

Mice dosed with antibody in the presence of vehicle (Group C) were foundto exhibit normal cognitive performances with a mean discriminationindex of 0.439±0.049 (FIG. 6A). These mice were not significantlydifferent from vehicle control mice (p=0.9163) and significantlydifferent from AβO injected mice (p<0.0001).

When co-injected with AβO, the antibody fully prevented AβO-inducedcognitive deficits in the NOR test. Indeed, mice from Group D exhibiteda mean discrimination index of 0.481±0.055, not different from controlmice (p=0.6126) but different from AβO-injected mice (p=0.0002) (FIG.6A). Taken together, the data suggest that antibody 301-17 offeredprotection against AβO-induced cognitive deficits.

The NOR assay was repeated with the recombinant 301-17 comprising amouse IgG1 backbone. The concentration of antibody used was however wasless than the previously described experiment with the oligomer:antibody ratio being only 1.62. As shown in FIG. 6D, when co-injectedwith AβO, the lower ratio of antibody was still capable of fullypreventing AβO-induced cognitive deficits in the NOR test.

Example 12

The novel object recognition test described in Example 10 was used totest a recombinant 305-61 antibody (mouse IgG1) (parent monoclonalraised using cyclo(CGQKLVG)). The following treatment schedule was used.

GROUP Treatment ICV N A Vehicle Vehicle 12 B Vehicle AbO 12 C Antibody(QKLV) Vehicle 12 D Antibody (QKLV) AbO 12

As described in Example 10, 24 animals were injected per day. In thepresent assay, before ICV injection, 4 μL of antibody 1 were incubatedfor 30 minutes at room temperature with 1 μL vehicle (i.e. buffer for Aβoligomerization) or 1 μL AβO (50 pmoles) corresponding to anantibody/AμO molar ratio of 5:1.

Results

Mice from the vehicle control group (Group A) exhibited normal behaviorwith a mean discrimination index of 0.39±0.05 (FIG. 6B). These resultsare in agreement with previous observations of similar control groups.As expected, a single ICV injection of AβO (Group B) resulted in asignificant impairment (p+=0.0127) of the cognitive performance whencompared to vehicle control mice; with a mean discrimination index of0.05±0.09. AβO-injected mice were not able to discriminate between noveland familiar objects (FIG. 6B).

Mice dosed with antibody in the presence of vehicle (Group C) were foundto exhibit normal cognitive performances with a mean discriminationindex of 0.36±0.05 (FIG. 6B). These mice were not significantlydifferent from vehicle control mice and were significantly differentfrom AβO injected mice.

When co-injected with AβO, antibody 305-61 fully prevented AβO-inducedcognitive deficits in the NOR test. Indeed, mice from Group D exhibiteda mean discrimination index of 0.38±0.06, not different from controlmice but different from AβO-injected mice (p=0.0135) (FIG. 6B). The datasuggest this antibody offered protection against AβO-induced cognitivedeficits.

Example 13

The novel object recognition test described in Example 10 was used totest an recombinant antibody (303-25, IgG1). The following treatmentschedule was used.

GROUP Treatment ICV N A Vehicle Vehicle 12 B Vehicle AbO 12 C Antibody(HDSG) Ab1 Vehicle 12 D Antibody (HDSG) AbO 12 E Isotype control Ab2Vehicle 12 F Isotype control AbO 12

An antibody:oligomer ratio of 5:1 was used. As described in Example 10,24 animals were injected per day, ICV injection took place on day 0, thebehavioural assay was conducted on day 7 or 8, and brains collected onday 10 for ELISA on hippocampal markers.

Results

Mice from the vehicle control group (Group A) exhibited normal behaviorwith a mean discrimination index of 0.29±0.03 (FIG. 6C). These resultsare in agreement with previous observations of similar control groups atSynAging. As expected, a single icy injection of AβO (Group B) resultedin a significant impairment (p=0.0150) of the cognitive performance whencompared to vehicle control mice; with a mean discrimination index of−0.08±0.1, AβO-injected mice were not able to discriminate between noveland familiar objects (FIG. 6C).

Mice dosed with the recombinant 303-25 antibody in the presence ofvehicle (Group C) were found to exhibit normal cognitive performanceswith a mean discrimination index of 0.35±0.06 (FIG. 6C). These mice werenot different from vehicle control mice (p=0.4871) and significantlydifferent from AβO injected mice (p=0.0047).

Similarly, mice dosed with antibody 2 (isotype control) in the presenceof vehicle (Group E) were found to exhibit normal cognitive performanceswith a mean discrimination index of 0.29±0.05 (FIG. 6C). These mice werenot different from vehicle control mice (p=0.9578) and significantlydifferent from AβO injected mice (p=0.0121).

When co-injected with AβO, test antibody prevented AβO-induced cognitivedeficits in the NOR test. Indeed, mice from Group D exhibited a meandiscrimination index of 0.30±0.08, not different from control mice(p=0.6715) but different from AβO-injected mice (p=0.0120) (FIG. 6C).

When co-injected with AβO, antibody 2 (isotype control) failed toprevent AβO-induced cognitive deficits in the NOR test. Indeed, micefrom Group F exhibited a mean discrimination index of 0.080±0.079, notdifferent from AβO-injected mice (p=0.1962), and lower but notstatistically different from control mice (p=0.1176) (FIG. 6C).

Example 14

Brains were collected from the same mice that underwent the behavioraltesting in Examples 11-13. The hippocampus (relevant structure formemory formation) was dissected and homogenized in RIPA buffercontaining an anti-protease cocktail. The tissue was lysed by 3 freezethaw cycles carried out in liquid nitrogen and a water bath at 37C andthe supernatants were recovered after centrifuging.

The lysate was analyzed for levels of TNF-alpha (increases withinflammation) and levels of the synaptic markers PSD-95 and SNAP-25(which go down when there is synaptic damage) using commercial ELISAassays. Data are represented as mean±SEM. Data are expressed as pgprotein per μg total protein. Statistical significance between groupsare evaluated and considered statistically different to another groupwhen p<0.05 (*: different from vehicle control mice, and #: differentfrom AβO-injected mice).

303-25

As expected, mice ICV injected with AβO oligomers exhibited a decreasedlevel of hippocampal PSD-95 (4.01±0.14 pg/μg total protein)statistically different from control mice (p<0.0001) (FIG. 7D).

Mice dosed with antibody 301-25 (antibody 1) or isotype control(antibody 2) in the presence of vehicle exhibited a normal level, notdifferent from control mice, of hippocampal PSD-95 of 5.02±0.14 and4.98±0.08 pg/μg total protein for antibody 1 and 2, respectively (FIG.7D).

When co-injected with AβO, antibody 1 significantly improved PSD-95level. Mice from Group D exhibited a level of hippocampal PSD-95 of4.79±0.09 pg/μg total protein, different from control mice (p=0.0016)but also different from AβO-injected mice (p=0.0003) (FIG. 7D).

When co-injected with AβO, antibody 2 failed to improve hippocampalPSD-95 level. Mice from Group F exhibited a PSD-95 concentration of4.34±0.11 pg/μg total protein, significantly lower than control mice(p<0.0001) and not different from AβO-injected mice (p=0.1189) (FIG.7D).

As expected, mice ICV injected with AβO oligomers exhibited a decreasedlevel of hippocampal SNAP25 (9.36±0.34 pg/μg total protein) differentfrom control mice (p<0.0001) (FIG. 7E).

Mice dosed with antibody 1 or antibody 2 in the presence of vehicleexhibited a normal level of hippocampal SNAP25 of 13.93±0.57 and12.94±0.26 pg/μg total protein for antibody 1 and antibody 2,respectively (FIG. 7E). These values were not different from controlmice (p=0.8399) for antibody 1 but statistically different from controlmice (p=0.0015) for antibody 2.

As expected, mice ICV injected with AβO oligomers exhibited an increasedlevel of hippocampal TNFα (2.39±0.14 pg/μg total protein) different fromcontrol mice (p<0.0001) (FIG. 7F).

Mice dosed with antibody 1 in the presence of vehicle exhibited a normallevel of hippocampal TNFα of 0.95±0.1 pg/μg total protein (FIG. 7F). Incontrast and for unknown reason, mice dosed with antibody 2 in thepresence of vehicle exhibited an increased level of hippocampal TNFα of1.69±0.07 pg/μg total protein, as compared to control mice (p<0.0001).

The presence antibody 1 resulted in a decreased level of TNFα in miceco-injected with AβO (FIG. 7F). Mice from group D exhibited a level ofhippocampal TNFα of 1.20±0.10 pg/μg total protein, not different fromcontrol mice (p=0.908) and different from AβO-injected mice (p<0.0001).When co-injected with AβO, antibody 2 failed to inhibit the AβO-inducedincrease of TNFα with a level of hippocampal TNFα of 2.16±0.10 pg/μgtotal protein (p<0.0001 as compared to control and p=0.2481 as comparedto AβO-injected mice).

305-61

Similar results were seen with 305-61. The 305-61 antibody showedcomplete protection in the behavioral assay and also showedstatistically significant improvement in both AbO induced SNAP25 (FIG.7B) and PSD-95 level changes (FIG. 7A). The antibody alone had noeffect.

The antibody also significantly decreased levels of TNF-alpha induced byAbO, as shown in FIG. 7C.

301-17

Both NOR experiments using 301-17 antibodies (e.g. monoclonal and IgG1recombinant) showed complete protection in the behavioral assay. Thebrain of the animals treated with or without recombinant 301-17 weretested PSD-95, SNAP25 and TNF-alpha levels. The antibody alsosignificantly improved both SNAP25 (FIG. 7H) and PSD-95 levels (FIG.7G). There was a trend for reduced TNF-alpha levels.

Example 15 Recombinant Antibodies (HHQK)

Recombinant IgG1 and IgG2a 301-17 constructs were made by grafting thevariable region of hybridoma-derived 301-17 onto a murine IgG1 or IgG2abackbone (WuXi, Biologics).

The 301-17 IgG1 and IgG2a antibodies were tested and compared to theparent hybridoma-purified IgG3 antibody for binding characteristics asdescribed below.

301-17 IgG2a ProteOn Biosensor (BioRad) Binding to AbO:

Recombinant 301-17 IgG2a and hybridoma-purified 301-17 IgG3 werecaptured with anti-mouse IgG or amine coupling on Proteon GLM Sensorchips and tested for AbO binding (SynAging AbO). AbO 3 fold-dilutionswere used: 1 uM, 0.33 uM, 0.11 uM, 37 nM, 12.3 nM. Assay buffer wasPBS-E+Tween 20+2 mg/ml BSA.

Results:

Approximate kinetic values were:

Hybridoma: KD=26.9 nM

IgG2a-301-17 antibody: KD=16.2-19.5 nM

No binding was detected with control mouse IgG.

Recombinant 301-17 IgG1 had a similar KD to the IgG2a recombinant.

301-17 IgG2a ProteOn Biosensor (BioRad) Binding to Cyclic PeptideEpitope:

Recombinant 301-17 IgG2a was amine-coupled to Proteon GLH biosensor chipand tested for binding to cyclopeptide of SEQ ID NO: 2 coupled to BSA.Cyclo-BSA 3-fold dilutions were used from 9 nM to 111 μM. Assay bufferwas PBS-E+0.05% Tween+10 mg/ml BSA. Antibody 301-17 IgG2a was found tobind cyclic peptide (SEQ ID NO: 2) conjugated to BSA with an approximateKD of 17 μM (average of 3 tests). No or negligible binding was detectedfor other commercial A-beta antibodies tested (pan-Abeta 6E10,Biolegend) and rabbit anti-A-beta antibodies (D54D2, Cell Signaling;ab201060, (abeam; NBP1-78007, Novus).

301-17 IgG1 MAAS-2 Binding to AbO:

Recombinant 301-17 IgG1 and hybridoma-purified 301-17 IgG3 wereimmobilized on MAAS-2 sensor chips and tested for binding to AbO(SynAging) at 1 uM. Under the conditions tested, the recombinant IgG1301-17 antibody gave a greater signal than the hybridoma-purifiedantibody in 2 tests (40-55 BRU vs 15-25 BRU, respectively). Little or nobinding was detected with control mouse IgG.

301-17 IgG1 MAAS-2 Binding to Cyclic Peptide Epitope:

Recombinant 301-17 IgG1 was immobilized on MAAS-2 sensor chip and testedfor binding to cyclopeptide of SEQ ID NO: 2 coupled to BSA at pH 6.5,7.5 or 8.0. Equivalently high levels of binding were observed for 301-17IgG1 under all 3 pH conditions (˜400 BRUs). Little or no binding wasdetected under any of the pH conditions for control mouse IgG or thepan-Abeta 6E10 antibody (Biolegend)

Example 16 Humanized Antibodies (HHQK)

Humanized IgG4 antibody constructs were prepared for 301-17 andsequenced (Abzena Cambridge UK).

Briefly RNA was extracted from the hybridoma 301-17 cell pellet using anRNeasy Mini Kit (Qiagen, Hilden, Germany). V-regions were amplified byRT-PCR using degenerate primer pools for murine antibody signalsequences together with constant region primers for each of IgG and Igκ.Heavy chain V region mRNA was amplified using a set of six degenerateprimer pools (A to F) specific for VH signal sequences together withIgG-specific constant region primers. The light chain V region mRNA wasamplified using a set of eight signal sequence-specific degenerateprimer pools, seven for the kappa cluster (Igκ-A to Igκ-G) and one forthe lambda cluster (IgA), together with K or A constant region primers.The PCR products obtained were purified, cloned into a ‘TA’ cloningvector (pGEM-T Easy, Promega, Madison, USA), transformed into E. coliand individual colonies sequenced.

Chimeric constructs (V0H0 and V0k0) were prepared using the variableregions from the hybridoma which were cloned into a human IgG4framework. The chimeric constructs were then humanized to create 6humanized heavy chains (VH1-6) and 6 light chains (Vk1-6). VH1-6 andVk4-6 constructs were mixed to create different humanized antibodiese.g. VH2Vk4.

The fully humanized antibodies were prepared using Composite HumanAntibody™ technology. The designed variable region genes were clonedinto vectors encoding a human IgG4 (S241 P) heavy chain constant domainand a human kappa light chain constant domain. Chimeric and humanizedantibodies were transiently expressed in CHO cells and Protein Apurified and tested. All 301-17 humanized antibodies selectively boundSEQ ID NO: 2 BSA with binding affinities within 2-fold of the referencechimeric antibody. Binding was determined using single cycle Biacoreanalysis. Antibodies were analysed in two separate experiments.

Humanized antibody sequences are provided in Table 13 and 14 (301-17).The CDR sequences of each antibody sequences are bolded and underlined.The CDRs of 301-11 or any other antibody described herein can be used toreplace the CDRs in the humanized constructs as shown for example inTable 12.

TABLE 12 Variable domain of humanized antibodies Chimeric/HumanizedAntibody 301-11 cDNA Sequence Polypeptide sequence VH0*CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGG QVQLQQPGAELVKPGA SEQ IDNO: GCTTCAGTGAAGATGTCCTGCAAGGCTTCT GGATTCACTTTCAGT SVKMSCKAS GFTFSDY125, 126 GACTATTAC ATAAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTT YINWVKQRPGQGLEWI Chimeric GAGTGGATTGGAGAT ATTAGTGATGGTGGTAGTTACACC TACAATGD ISDGGSYT YNAKFK GCTAAGTTCAAGAGCAAGGCCACACTGACTCTGGACACATCCTCCSKATLTLDTSSSTAYM AGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTQLSSLTSEDSAVYYC A GCGGTCTATTACTGT GCAAGAGATTACTACGGTAGTAGTAGCTACRDYYGSSSYTSGFAY W ACCTCGGGCTTTGCTTAC TGGGGCGCAGGCACCACGGTCACCGTCGAGTTVTVSS TCCTCA VH1 CAGGTCCAACTGGTGCAGTCTGGGGCTGAGCTTAAGAAGCCTGGGQVQLVQSGAELKKPGA SEQ ID NO: GCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATTCACTTTCAGT SVKMSCKAS GFTFSDY 127, 128 GACTATTACATAAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTT Y INWVKQRPGQGLEWI GAGTGGATTGGAGATATTAGTGATGGTGGTAGTTACACC TACAAT GD ISDGGSYT YNAKFKGCTAAGTTCAAGAGCAGAGCCACACTGACTCTGGACACATCCATA SRATLTLDTSISTAYMAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCT QLSSLTSEDSAVYYC AGCGGTCTATTACTGT GCAAGAGATTACTACGGTAGTAGTAGCTAC RDYYGSSSYTSGFAY WACCTCGGGCTTTGCTTAC TGGGGCCAAGGCACCACGGTCACCGTC GQGTTVTVSS TCCTCA VH2CAGGTCCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGG QVQLVQSGAEVKKPGA SEQ IDNO: GCTTCAGTGAAGATGTCCTGCAAGGCTTCT GGATTCACTTTCAGT SVKMSCKAS GFTFSDY129, 130 GACTATTAC ATAAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTT YINWVKQRPGQGLEWI GAGTGGATTGGAGAT ATTAGTGATGGTGGTAGTTACACC TACAAT GDISDGGSYT YNAKFK GCTAAGTTCAAGAGCAGAGCCACACTGACTCTGGACACATCCATASRATLTLDTSISTAYM AGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGACACGELSSLRSEDTAVYYC A GCGGTCTATTACTGT GCAAGAGATTACTACGGTAGTAGTAGCTACRDYYGSSSYTSGFAY W ACCTCGGGCTTTGCTTAC TGGGGCCAAGGCACCACGGTCACCGTCGQGTTVTVSS TCCTCA VH3 CAGGTCCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGQVQLVQSGAEVKKPGA SEQ ID NO: GCTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATTCACTTTCAGT SVKVSCKAS GFTFSDY 131, 132 GACTATTACATAAACTGGGTGCGACAGAGGCCTGGACAAGGCCTT Y INWVRQRPGQGLEWI GAGTGGATTGGAGATATTAGTGATGGTGGTAGTTACACC TACAAT GD ISDGGSYT YNAKFKGCTAAGTTCAAGAGCAGAGCCACACTGACTCTGGACACATCCATA SRATLTLDTSISTAYMAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGACACG ELSSLRSEDTAVYYC AGCGGTCTATTACTGT GCAAGAGATTACTACGGTAGTAGTAGCTAC RDYYGSSSYTSGFAY WACCTCGGGCTTTGCTTAC TGGGGCCAAGGCACCACGGTCACCGTC GQGTTVTVSS TCCTCA VH4CAGGTCCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGG QVQLVQSGAEVKKPGA SEQ IDNO: GCTTCAGTGAAGGTGTCCTGCAAGGCTTCT GGATTCACTTTCAGT SVKVSCKAS GFTFSDY133, 134 GACTATTAC ATAAACTGGGTGCGACAGAGGCCTGGACAAGGCCTT YINWVRQRPGQGLEWI GAGTGGATTGGAGAT ATTAGTGATGGTGGTAGTTACACC TACAAT GDISDGGSYT YNAKFK GCTAAGTTCAAGAGCAGAGTCACACTGACTCTGGACACATCCATASRVTLTLDTSISTAYM AGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGACACGELSSLRSEDTAVYYC A GCGGTCTATTACTGT GCAAGAGATTACTACGGTAGTAGTAGCTACRDYYGSSSYTSGFAY W ACCTCGGGCTTTGCTTAC TGGGGCCAAGGCACCACGGTCACCGTCGQGTTVTVSS TCCTCA VH5 CAGGTCCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGQVQLVQSGAEVKKPGA SEQ ID NO: GCTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATTCACTTTCAGT SVKVSCKAS GFTFSDY 135, 136 GACTATTACATAAACTGGGTGCGACAGAGGCCTGGACAAGGCCTT Y INWVRQRPGQGLEWM GAGTGGATGGGAGATATTAGTGATGGTGGTAGTTACACC TACAAT GD ISDGGSYT YNAKFKGCTAAGTTCAAGAGCAGAGTCACACTGACTAGGGACACATCCATA SRVTLTRDTSISTAYMAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGACACG ELSSLRSEDTAVYYC AGCGGTCTATTACTGT GCAAGAGATTACTACGGTAGTAGTAGCTAC RDYYGSSSYTSGFAY WACCTCGGGCTTTGCTTAC TGGGGCCAAGGCACCACGGTCACCGTC GQGTTVTVSS TCCTCA VH6CAGGTCCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGG QVQLVQSGAEVKKPGA SEQ IDNO: GCTTCAGTGAAGGTGTCCTGCAAGGCTTCT GGATTCACTTTCAGT SVKVSCKAS GFTFSDY137, 138 GACTATTAC ATAAACTGGGTGCGACAGAGGCCTGGACAAGGCCTT YINWVRQRPGQGLEWM GAGTGGATGGGAGAT ATTAGTGATGGTGGTAGTTACACC TACAAT GDISDGGSYT YNAKFQ GCTAAGTTCCAGGGCAGAGTCACAATGACTAGGGACACATCCATAGRVTMTRDTSISTAYM AGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGACACGELSSLRSEDTAVYYC A GCGGTCTATTACTGT GCAAGAGATTACTACGGTAGTAGTAGCTACRDYYGSSSYTSGFAY W ACCTCGGGCTTTGCTTAC TGGGGCCAAGGCACCACGGTCACCGTCGQGTTVTVSS TCCTCA VK0* GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTDVLMTQTPLSLPVSLG SEQ ID NO: GGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGTCTGCTC DQASISCRSS QSLLNS 139, 140 AACAGTAGAACCCGAAAGAACTACTTAGAATGGTACCTGCAGAAA RTRKNY LEWYLQKPGQ chimericCCAGGCCAGTCTCCAAAGCTCCTGATCTAC TGGGCATCC AACCGA SPKLLIY WAS NRFSGVTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACA PDRFSGSGSGTDFTLKGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGA ISRVEAEDLGVYYC KQGTTTATTACTGC AAGCAATCTTATAATCTGTACACG TTTGGCAGC SYNLYT FGSGTKLEIKGGGACCAAGCTGGAGATCAAA VK1 GATGTTTTGATGACCCAATCTCCACTCTCCCTGCCTGTCACCCTTDVLMTQSPLSLPVTLG SEQ ID NO: GGACAGCCGGCCTCCATCTCTTGCAGATCTAGTCAGAGTCTGCTC QPASISCRSS QSLLNS 141, 142 AACAGTAGAACCCGAAAGAACTACTTAGAATGGTTTCAGCAGAAA RTRKNY LEWFQQKPGQ CCAGGCCAGTCTCCAAGGCGCCTGATCTACTGGGCATCC AACCGA SPRRLIY WAS NRFSGVTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACA PDRFSGSGSGTDFTLKGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTTGGA ISRVEAEDVGVYYC KQGTTTATTACTGC AAGCAATCTTATAATCTGTACACG TTTGGCCAA SYNLYT FGQGTKLEIKGGGACCAAGCTGGAGATCAAA VK2 GATGTTGTGATGACCCAATCTCCACTCTCCCTGCCTGTCACCCTTDVVMTQSPLSLPVTLG SEQ ID NO: GGACAGCCGGCCTCCATCTCTTGCAGATCTAGTCAGAGTCTGCTC QPASISCRSS QSLLNS 143, 144 AACAGTAGAACCCGAAAGAACTACTTAGAATGGTTTCAGCAGAAA RTRKNY LEWFQQKPGQ CCAGGCCAGTCTCCAAGGCGCCTGATCTACTGGGCATCC AACCGA SPRRLIY WAS NRFSGVTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACA PDRFSGSGSGTDFTLKGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTTGGA ISRVEAEDVGVYYC KQGTTTATTACTGC AAGCAATCTTATAATCTGTACACG TTTGGCCAA SYNLYT FGQGTKLEIKGGGACCAAGCTGGAGATCAAA VK3 GATGTTGTGATGACCCAATCTCCACTCTCCCTGCCTGTCACCCTTDVVMTQSPLSLPVTLG SEQ ID NO: GGACAGCCGGCCTCCATCTCTTGCAGATCTAGTCAGAGTCTGCTC QPASISCRSS QSLLNS 145, 146 AACAGTAGAACCCGAAAGAACTACTTAGAATGGTTTCAGCAGAGG RTRKNY LEWFQQRPGQ CCAGGCCAGTCTCCAAGGCGCCTGATCTACTGGGCATCC AACCGA SPRRLIY WAS NRFSGVTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACA PDRFSGSGSGTDFTLKGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTTGGA ISRVEAEDVGVYYC KQGTTTATTACTGC AAGCAATCTTATAATCTGTACACG TTTGGCCAA SYNLYT FGQGTKLEIKGGGACCAAGCTGGAGATCAAA VK4 GATGTTCTGATGACCCAATCTCCACTCTCCCTGCCTGTCACCCTTDVLMTQSPLSLPVTLG SEQ ID NO: GGACAGCCGGCCTCCATCTCTTGCAGATCTAGTCAGAGTCTGCTC QPASISCRSS QSLLNS 147, 148 AACAGTAGAACCCGAAAGAACTACTTAGAATGGTACCTGCAGAGG RTRKNY LEWYLQRPGQ CCAGGCCAGTCTCCAAAGCTGCTGATCTACTGGGCATCC AACCGA SPKLLIY WAS NRFSGVTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACA PDRFSGSGSGTDFTLKGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTTGGA ISRVEAEDVGVYYC KQGTTTATTACTGC AAGCAATCTTATAATCTGTACACG TTTGGCCAA SYNLYT FGQGTKLEIKGGGACCAAGCTGGAGATCAAA VK5 GATGTTCTGATGACCCAATCTCCACTCTCCCTGCCTGTCACCCTTDVLMTQSPLSLPVTLG SEQ ID NO: GGACAGCCGGCCTCCATCTCTTGCAGATCTAGTCAGAGTCTGCTC QPASISCRSS QSLLNS 149, 150 AACAGTAGAACCCGAAAGAACTACTTAGAATGGTACCAGCAGAGG RTRKNY LEWYQQRPGQ CCAGGCCAGTCTCCAAGGCTGCTGATCTACTGGGCATCC AACCGA SPRLLIY WAS NRFSGVTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACA PDRFSGSGSGTDFTLKGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTTGGA ISRVEAEDVGVYYC KQGTTTATTACTGC AAGCAATCTTATAATCTGTACACG TTTGGCCAA SYNLYT FGQGTKLEIKGGGACCAAGCTGGAGATCAAA VK6 GATGTTGTGATGACCCAATCTCCACTCTCCCTGCCTGTCACCCTTDVVMTQSPLSLPVTLG SEQ ID NO: GGACAGCCGGCCTCCATCTCTTGCAGATCTAGTCAGAGTCTGCTC QPASISCRSS QSLLNS 151, 152 AACAGTAGAACCCGAAAGAACTACTTAGAATGGTACCAGCAGAGG RTRKNY LEWYQQRPGQ CCAGGCCAGTCTCCAAGGCTGCTGATCTACTGGGCATCC AACCGA SPRLLIY WAS NRFSGVTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACA PDRFSGSGSGTDFTLKGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTTGGA ISRVEAEDVGVYYC KQGTTTATTACTGC AAGCAATCTTATAATCTGTACACG TTTGGCCAA SYNLYT FGQGTKLEIKGGGACCAAGCTGGAGATCAAA *VH0 and VK0 denotes chimeric antibodies comprisedof the human constant domain and mouse variable domain sequences and areprovided for comparison.

TABLE 13 Variable domain of humanized antibodies Chimeric/HumanizedPolypeptide Antibody cDNA Sequence Sequence 301-17CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGG QVQLQQPGAELVKPGA VH0*GCTTCAGTGAAGATGTCCTGCAAGGCTTCT GGCTACAGCTTCACC SVKMSCKAS GYSFTSY SEQ IDNO: AGCTACTGG ATAAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTT W INWVKQRPGQGLEWI153, 154 GAGTGGATTGGAGAT GTGCATCCTGGTAGAGGCGTGTCCACA TAC GD VHPGRGVSTYNAKF (chimeric) AATGCTAAGTTCAAGAGCAAGGCCACACTGACTCTGGACACATCCKSKATLTLDTSSSTAY TCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACMQLSSLTSEDSAVYYC TCTGCGGTCTATTACTGT AGCAGATCCCATGGTAACACCTACTGGSRSHGNTYWFFDV WGA TTTTTTGACGTC TGGGGCGCAGGCACCACGGTCACCGTCTCCTCAGTTVTVSS VH1 CAGGTCCAACTGGTGCAGTCTGGGGCTGAGCTTAAGAAGCCTGGGQVQLVQSGAELKKPGA SEQ ID NO: GCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACAGCTTCACC SVKMSCKAS GYSFTSY 155, 156 AGCTACTGGATAAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTT W INWVKQRPGQGLEWI GAGTGGATTGGAGATGTGCATCCTGGTAGAGGCGTGTCCACA TAC GD VHPGRGVST YNAKFAATGCTAAGTTCAAGAGCAGAGCCACACTGACTCTGGACACATCC KSRATLTLDTSISTAYATAAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGAC MQLSSLTSEDSAVYYCTCTGCGGTCTATTACTGT AGCAGATCCCATGGTAACACCTACTGG SRSHGNTYWFFDV WGQTTTTTTGACGTC TGGGGCCAAGGCACCACGGTCACCGTCTCCTCA GTTVTVSS VH2CAGGTCCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGG QVQLVQSGAEVKKPGA SEQ IDNO: GCTTCAGTGAAGATGTCCTGCAAGGCTTCT GGCTACAGCTTCACC SVKMSCKAS GYSFTSY157, 158 AGCTACTGG ATAAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTT WINWVKQRPGQGLEWI GAGTGGATTGGAGAT GTGCATCCTGGTAGAGGCGTGTCCACA TAC GDVHPGRGVST YNAKF AATGCTAAGTTCAAGAGCAGAGCCACACTGACTCTGGACACATCCKSRATLTLDTSISTAY ATAAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGACMELSSLRSEDTAVYYC ACGGCGGTCTATTACTGT AGCAGATCCCATGGTAACACCTACTGGSRSHGNTYWFFDV WGQ TTTTTTGACGTC TGGGGCCAAGGCACCACGGTCACCGTCTCCTCAGTTVTVSS VH3 CAGGTCCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGQVQLVQSGAEVKKPGA SEQ ID NO: GCTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGCTACAGCTTCACC SVKVSCKAS GYSFTSY 159, 160 AGCTACTGGATAAACTGGGTGCGACAGAGGCCTGGACAAGGCCTT W INWVRQRPGQGLEWI GAGTGGATTGGAGATGTGCATCCTGGTAGAGGCGTGTCCACA TAC GD VHPGRGVST YNAKFAATGCTAAGTTCAAGAGCAGAGCCACACTGACTCTGGACACATCC KSRATLTLDTSISTAYATAAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGAC MELSSLRSEDTAVYYCACGGCGGTCTATTACTGT AGCAGATCCCATGGTAACACCTACTGG SRSHGNTYWFFDV WGQTTTTTTGACGTC TGGGGCCAAGGCACCACGGTCACCGTCTCCTCA GTTVTVSS VH4CAGGTCCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGG QVQLVQSGAEVKKPGA SEQ IDNO: GCTTCAGTGAAGGTGTCCTGCAAGGCTTCT GGCTACAGCTTCACC SVKVSCKAS GYSFTSY161, 162 AGCTACTGG ATAAACTGGGTGCGACAGAGGCCTGGACAAGGCCTT WINWVRQRPGQGLEWI GAGTGGATTGGAGAT GTGCATCCTGGTAGAGGCGTGTCCACA TAC GDVHPGRGVST YNAKF AATGCTAAGTTCAAGAGCAGAGTCACACTGACTCTGGACACATCCKSRVTLTLDTSISTAY ATAAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGACMELSSLRSEDTAVYYC ACGGCGGTCTATTACTGT AGCAGATCCCATGGTAACACCTACTGGSRSHGNTYWFFDV WGQ TTTTTTGACGTC TGGGGCCAAGGCACCACGGTCACCGTCTCCTCAGTTVTVSS VH5 CAGGTCCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGQVQLVQSGAEVKKPGA SEQ ID NO: GCTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGCTACAGCTTCACC SVKVSCKAS GYSFTSY 163, 164 AGCTACTGGATAAACTGGGTGCGACAGAGGCCTGGACAAGGCCTT W INWVRQRPGQGLEWM GAGTGGATGGGAGATGTGCATCCTGGTAGAGGCGTGTCCACA TAC GD VHPGRGVST YNAKFAATGCTAAGTTCAAGAGCAGAGTCACACTGACTAGGGACACATCC KSRVTLTRDTSISTAYATAAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGAC MELSSLRSEDTAVYYCACGGCGGTCTATTACTGT AGCAGATCCCATGGTAACACCTACTGG SRSHGNTYWFFDV WGQTTTTTTGACGTC TGGGGCCAAGGCACCACGGTCACCGTCTCCTCA GTTVTVSS VH6CAGGTCCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGG QVQLVQSGAEVKKPGA SEQ IDNO: GCTTCAGTGAAGGTGTCCTGCAAGGCTTCT GGCTACAGCTTCACC SVKVSCKAS GYSFTSY165, 166 AGCTACTGG ATAAACTGGGTGCGACAGAGGCCTGGACAAGGCCTT WINWVRQRPGQGLEWM GAGTGGATGGGAGAT GTGCATCCTGGTAGAGGCGTGTCCACA TAC GDVHPGRGVST YNAKF AATGCTAAGTTCCAGGGCAGAGTCACAATGACTAGGGACACATCCQGRVTMTRDTSISTAY ATAAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGACMELSSLRSEDTAVYYC ACGGCGGTCTATTACTGT AGCAGATCCCATGGTAACACCTACTGGSRSHGNTYWFFDV WGQ TTTTTTGACGTC TGGGGCCAAGGCACCACGGTCACCGTCTCCTCAGTTVTVSS VK0* GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTDVLMTQTPLSLPVSLG SEQ ID NO: GGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTA DQASISCRSS QSIVHS 167, 168 CATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCA NGNTY LEWYLQKPGQS (Chimeric)GGCCAGTCTCCAAAGCTCCTGATCTAC AAAGTTTCC AACCGATTT PKLLIY KVS NRFSGVPTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT DRFSGSGSGTDFTLKITTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTT SRVEAEDLGVYYC FQGTATTACTGC TTTCAAGGTTCACATGTTCCTTTCACT TTTGGCAGC SHVPFT FGSGTKLEIKGGGACCAAGCTGGAGATCAAA VK1 GATGTTTTGATGACCCAATCTCCACTCTCCCTGCCTGTCACCCTTDVLMTQSPLSLPVTLG SEQ ID NO: GGACAGCCGGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTA QPASISCRSS QSIVHS 169, 170 CATAGTAATGGAAACACCTATTTAGAATGGTTTCAGCAGAAACCA NGNTY LEWFQQKPGQS GGCCAGTCTCCAAGGCGCCTGATCTACAAAGTTTCC AACCGATTT PRRLIY KVS NRFSGVPTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT DRFSGSGSGTDFTLKITTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTTGGAGTT SRVEAEDVGVYYC FQGTATTACTGC TTTCAAGGTTCACATGTTCCTTTCACT TTTGGCCAA SHVPFT FGQGTKLEIKGGGACCAAGCTGGAGATCAAA VK2 GATGTTGTGATGACCCAATCTCCACTCTCCCTGCCTGTCACCCTTDVVMTQSPLSLPVTLG SEQ ID NO: GGACAGCCGGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTA QPASISCRSS QSIVHS 171-172 CATAGTAATGGAAACACCTATTTAGAATGGTTTCAGCAGAAACCA NGNTY LEWFQQKPGQS GGCCAGTCTCCAAGGCGCCTGATCTACAAAGTTTCC AACCGATTT PRRLIY KVS NRFSGVPTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT DRFSGSGSGTDFTLKITTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTTGGAGTT SRVEAEDVGVYYC FQGTATTACTGC TTTCAAGGTTCACATGTTCCTTTCACT TTTGGCCAA SHVPFT FGQGTKLEIKGGGACCAAGCTGGAGATCAAA VK3 GATGTTGTGATGACCCAATCTCCACTCTCCCTGCCTGTCACCCTTDVVMTQSPLSLPVTLG SEQ ID NO: GGACAGCCGGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTA QPASISCRSS QSIVHS 173-174 CATAGTAATGGAAACACCTATTTAGAATGGTTTCAGCAGAGGCCA NGNTY LEWFQQRPGQS GGCCAGTCTCCAAGGCGCCTGATCTACAAAGTTTCC AACCGATTT PRRLIY KVS NRFSGVPTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT DRFSGSGSGTDFTLKITTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTTGGAGTT SRVEAEDVGVYYC FQGTATTACTGC TTTCAAGGTTCACATGTTCCTTTCACT TTTGGCCAA SHVPFT FGQGTKLEIKGGGACCAAGCTGGAGATCAAA VK4 GATGTTCTGATGACCCAATCTCCACTCTCCCTGCCTGTCACCCTTDVLMTQSPLSLPVTLG SEQ ID NO: GGACAGCCGGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTA QPASISCRSS QSIVHS 175-176 CATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAGGCCA NGNTY LEWYLQRPGQS GGCCAGTCTCCAAAGCTGCTGATCTACAAAGTTTCC AACCGATTT PKLLIY KVS NRFSGVPTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT DRFSGSGSGTDFTLKITTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTTGGAGTT SRVEAEDVGVYYC FQGTATTACTGC TTTCAAGGTTCACATGTTCCTTTCACT TTTGGCCAA SHVPFT FGQGTKLEIKGGGACCAAGCTGGAGATCAAA VK5 GATGTTCTGATGACCCAATCTCCACTCTCCCTGCCTGTCACCCTTDVLMTQSPLSLPVTLG SEQ ID NO: GGACAGCCGGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTA QPASISCRSS QSIVHS 177-178 CATAGTAATGGAAACACCTATTTAGAATGGTACCAGCAGAGGCCA NGNTY LEWYQQRPGQS GGCCAGTCTCCAAGGCTGCTGATCTACAAAGTTTCC AACCGATTT PRLLIY KVS NRFSGVPTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT DRFSGSGSGTDFTLKITTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTTGGAGTT SRVEAEDVGVYYC FQGTATTACTGC TTTCAAGGTTCACATGTTCCTTTCACT TTTGGCCAA SHVPFT FGQGTKLEIKGGGACCAAGCTGGAGATCAAA VK6 GATGTTGTGATGACCCAATCTCCACTCTCCCTGCCTGTCACCCTTDVVMTQSPLSLPVTLG SEQ ID NO: GGACAGCCGGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTA QPASISCRSS QSIVHS 179-180 CATAGTAATGGAAACACCTATTTAGAATGGTACCAGCAGAGGCCA NGNTY LEWYQQRPGQS GGCCAGTCTCCAAGGCTGCTGATCTACAAAGTTTCC AACCGATTT PRLLIY KVS NRFSGVPTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGAT DRFSGSGSGTDFTLKITTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATGTTGGAGTT SRVEAEDVGVYYC FQGTATTACTGC TTTCAAGGTTCACATGTTCCTTTCACT TTTGGCCAA SHVPFT FGQGTKLEIKGGGACCAAGCTGGAGATCAAA *VH0 and VK0 denotes chimeric antibodies comprisedof the human constant domain and mouse variable domain sequences and areprovided for comparison.

TABLE 14 Humanized antibody IgG4 sequence Constant regions cDNA SequencePolypeptide sequence IgG4 heavyGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCC ASTKGPSVFPLAPCSR chainAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAG STSESTAALGCLVKDY SEQ IDNO: GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCC FPEPVTVSWNSGALTS181-182 CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA GVHTFPAVLQSSGLYSGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC LSSVVTVPSSSLGTKTTTGGGCACGAAGACCTACACCTGCAATGTAGATCACAAGCCCAGC YTCNVDHKPSNTKVDKAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCA RVESKYGPPCPPCPAPTGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTC EFLGGPSVFLFPPKPKTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGG DTLMISRTPEVTCVVVACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGAC DVSQEDPEVQFNWYVDCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCAT GVEVHNAKTKPREEQFAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC NSTYRVVSVLTVLHQDCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAC WLNGKEYKCKVSNKGLGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCC PSSIEKTISKAKGQPRTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAG EPQVYTLPPSQEEMTKCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAG NQVSLTCLVKGFYPSDAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGC IAVEWESNGQPENNYKGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC TTPPVLDSDGSFFLYSTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC RLTVDKSRWQEGNVFSCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGG CSVMHEALHNHYTQKSAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC LSLSLGKTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA KappaCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT RTVAAPSVFIFPPSDE SEQ IDNO: GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT QLKSGTASVVCLLNNF183-184 AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC YPREAKVQWKVDNALQGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC SGNSQESVTEQDSKDSAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC TYSLSSTLTLSKADYEAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC KHKVYACEVTHQGLSSCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA PVTKSFNRGEC GAGTGTTAG

Example 17

A dot blot assay was used to detect antibody binding of antibodies tomonomeric, oligomeric and fibrillary A-beta. 10 ng of monomeric andfibril and 100 ng oligomeric A-beta was spotted on a PVDF membrane. Themembrane was blotted with antibodies 301-17, 303-25 and 305-62 as wellas prior art antibodies aducanumab, solanezumab and bapinezumab(Creative Biolabs). Antibodies 301-17 (FIG. 8 panel C), 303-25 (FIG. 8panel A) and 305-62 (FIG. 8 panel B) bound oligomeric but not monomericor fibrillar A-beta. Aducanumab exhibited preferential binding forfibrils, solanezumab exhibited preferential binding for monomers andbapinezumab showed significant binding to monomers, fibrils andoligomers.

Example 18

CDRs can also be identified using the Kabat numbering scheme. Forexample the CDRs for the humanized 301-17 constructs using the Kabatnumbering system are:

TABLE 15 Chain CDR Sequence SEQ ID NO. Heavy CDR-H1 SYWIN 195 CDR-H2*DVHPGRGVSTYNAKFKS* 196 CDR-H3 SHGNTYWFFDV 197 Light CDR-L1RSSQSIVHSNGNTYLE 198 CDR-L2 KVSNRFS 199 CDR-L3 FQGSHVPFT 200 *For VH6,CDR-H2sequence is DVHPGRGVSTYNAKFQG (SEQ ID NO. 201)

While the present application has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the application is not limited to the disclosedexamples. To the contrary, the application is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Specifically, the sequences associated with eachaccession numbers provided herein including for example accessionnumbers and/or biomarker sequences (e.g. protein and/or nucleic acid)provided in the Tables or elsewhere, are incorporated by reference inits entirely.

The scope of the claims should not be limited by the preferredembodiments and examples, but should be given the broadestinterpretation consistent with the description as a whole.

REFERENCES

-   [8] J. X. Lu, W. Qiang, W. M. Yau, C. D. Schwieters, S. C.    Meredith, R. Tycko, MOLECULAR STRUCTURE OF BETA-AMYLOID FIBRILS IN    ALZHEIMER'S DISEASE BRAIN TISSUE. CELL Vol. 154 p. 1257 (2013)[9] Y.    Xiao, B. MA, D. McElheny, S. Parthasarathy, F. Long, M. Hoshi, R.    Nussinov, Y. Ishii, A BETA (1-42) FIBRIL STRUCTURE ILLUMINATES    SELF-RECOGNITION AND REPLICATION OF AMYLOID IN ALZHEIMER'S DISEASE.    NAT. STRUCT. MOL. BIOL. Vol. 22 p. 499 (2015).-   [10] A. Petkova, W. Yau, R. Tycko EXPERIMENTAL CONSTRAINTS ON    QUATERNARY STRUCTURE IN ALZHEIMER'S BETA-AMYLOID FIBRILS    BIOCHEMISTRY V. 45 498 2006.-   [11] Giulian D, Haverkamp L J, Yu J, Karshin W, Tom D, Li J,    Kazanskaia A, Kirkpatrick J, Roher A E. The HHQK domain of β-amyloid    provides a structural basis for the immunopathology of Alzheimer's    disease, J. Biol. Chem. 1998, 273(45), 29719-26.-   [12] Winkler K, Scharnagl H, Tisljar U, Hoschutzky H, Friedrich I,    Hoffmann M M, Huttinger M, Wieland H, Marz W. Competition of Aβ    amyloid peptide and apolipoprotein E for receptor-mediated    endocytosis. J. Lipid Res. 1999, 40(3), 447-55.-   [16] SCIENTIFIC REPORTS |5: 9649| DOI: 10.1038/srep09649

1. A method of treating or preventing a disease or condition associatedwith and/or induced by soluble A-beta oligomer comprising administeringto a subject in need thereof: an isolated conformation specific and/orselective antibody or binding fragment thereof that specifically and/orselectively binds to a cyclic compound comprising an A-beta peptidehaving a sequence of QKL, HQK, KLV, HHQK (SEQ ID NO: 1), QKLV (SEQ IDNO: 5) or HDSG (SEQ ID NO: 9), they cyclic compound optionally having asequence of SEQ ID NO: 2, 3, 4, 6, 7, 8, 10, 11 or 12; an immunogencomprising a cyclic compound comprising an A-beta peptide having asequence of QKL, HQK, KLV, HHQK (SEQ ID NO: 1), QKLV (SEQ ID NO: 5) orHDSG (SEQ ID NO: 9); a cell expressing said antibody or binding fragmentthereof; or a nucleic acid encoding said antibody or binding fragmentthereof.
 2. The method of claim 1, wherein the cyclic compound that isspecifically and/or selectively bound has a sequence of SEQ ID NO: 2, 6or
 10. 3. The method of claim 1, wherein the antibody or bindingfragment selectively binds to the cyclic compound cover a correspondinglinear peptide and/or selectively binds A-beta oligomer over A-betamonomer and/or A-beta fibril, wherein the antibody is at least 2 fold, 3fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30fold, at least 40 fold, at least 50 fold, at least 100 fold, at least500 fold, at least 1000 fold more selective for the cyclic compound overthe corresponding linear compound.
 4. The method of claim 1 wherein theantibody or binding fragment thereof is a monoclonal antibody, achimeric antibody, a humanized antibody or a polyclonal antibody or abinding fragment of any of the foregoing.
 5. The method of claim 1,wherein the binding fragment is an antibody binding fragment selectedfrom Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers, nanobodies,minibodies, diabodies, and multimers thereof.
 6. The method of claim 1,wherein the antibody comprises a light chain variable region and a heavychain variable region, optionally fused, the heavy chain variable regioncomprising complementarity determining regions CDR-H1, CDR-H2 andCDR-H3, the light chain variable region comprising complementaritydetermining region CDR-L1, CDR-L2 and CDR-L3 and with the amino acidsequences of said CDRs comprising the sequences in Table 9 A to I. 7.The method of claim 1, wherein the antibody or binding fragmentcomprises a heavy chain variable region comprising: i) an amino acidsequence of a heavy chain variable sequence as set forth in Table 10, 12or 13; ii) an amino acid sequence with at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% sequence identity to said heavychain variable sequence set out in Table 10, 12 or 13, wherein the CDRsequences are the corresponding CDRs as set forth in Table 9, or iii) aconservatively substituted amino acid sequence i) wherein the CDRsequences are the corresponding CDRs as set forth in Table 9; andwherein the antibody comprises a light chain variable region comprisingi) an amino acid sequence of a light chain variable sequence as setforth in Table 10, 12 or 13, ii) an amino acid sequence with at least50%, at least 60%, at least 70%, at least 80%, at least 90% sequenceidentity to said light chain variable sequence as set out in Table 10,12 or 13, wherein the CDR sequences are the corresponding CDRs as setforth in Table 9, or iii) a conservatively substituted amino acidsequence i) wherein the CDR sequences a are the corresponding CDRs asset forth in Table
 9. 8. The method of claim 1, wherein the antibody orbinding fragment competes for binding to human A-beta with an antibodycomprising the CDR sequences as recited in Table 9 and/or antibodyproduced by the hybridoma cell line deposited under the provisions ofthe Budapest Treaty with the American Type Culture Collection (ATCC®)10801 University Blvd., Manassas, Va., 20110-2209, USA on Jul. 19, 2017and given the Accession number PTA-12431.
 9. The method of claim 1,wherein the antibody is comprised in an immunoconjugate comprising theantibody and a cytotoxic agent.
 10. The method of claim 1 wherein thecell expressing the antibody or binding fragment thereof or the nucleicacid molecule encoding the antibody of or binding fragment thereof isadministered, optionally wherein the nucleic acid molecule is comprisedin a vector.
 11. The method of claim 1, wherein the disease or conditionassociated with and/or induced by soluble A-beta oligomer is Alzheimer'sdisease (AD).
 12. The method of claim 1, wherein a combination ofantibodies or binding fragments is administered.
 13. A compositioncomprising two or more antibodies or binding fragments thereofcomprising a CDR set listed in Table 9, a variable heavy and lightdomain region combination listed in Table 10 or Table 12 or 13 or two ormore nucleic acid molecules encoding one of the foregoing.
 14. A methodof inhibiting A-beta oligomer propagation, the method comprisingcontacting a cell or tissue expressing A-beta with or administering to asubject in need thereof an effective amount of an isolated A-betaoligomer specific or selective antibody or binding fragment thereof thatspecifically and/or selectively binds to a cyclic compound comprising anA-beta peptide having a sequence of QKL, HQK, KLV, HHQK (SEQ ID NO: 1),QKLV (SEQ ID NO: 5) or HDSG (SEQ ID NO: 9), they cyclic compoundoptionally having a sequence of SEQ ID NO: 2, 3, 4, 6, 7, 8, 10, 11 or12; an immunogen comprising a cyclic compound comprising an A-betapeptide having a sequence of QKL, HQK, KLV, HHQK (SEQ ID NO: 1), QKLV(SEQ ID NO: 5) or HDSG (SEQ ID NO: 9); a cell expressing said antibodyor binding fragment thereof; or a nucleic acid encoding said antibody orbinding fragment thereof or immunoconjugate thereof, to inhibit A-betaaggregation and/or oligomer propagation.
 15. The method of claim 14,wherein a biological sample from the subject to be treated is assessedfor the presence or levels of A-beta using an antibody described herein.16. The method of claim 14, wherein more than one antibody orimmunoconjugate is administered.
 17. The method of claim 14, wherein theantibody or binding fragment thereof, immunoconjugate, cell or nucleicacid is administered directly to the brain or other portion of the CNS.18. A hybrdioma cell line deposited under the provisions of the BudapestTreaty with the American Type Culture Collection (ATCC®) 10801University Blvd., Manassas, Va., 20110-2209, USA on Jul. 19, 2017 andgiven the Accession number PTA-124318.
 19. An antibody or bindingfragment thereof comprising a light chain variable region and a heavychain variable region, optionally fused, the heavy chain variable regioncomprising complementarity determining regions CDR-H1, CDR-H2 andCDR-H3, the light chain variable region comprising complementaritydetermining region CDR-L1, CDR-L2 and CDR-L3 and with the amino acidsequences of said CDRs comprising the sequences in Table 9B, 9C, 9G, 9Hor 9I; or an antibody that competes for binding with said antibodycomprising a light chain variable region and a heavy chain variableregion, optionally fused, the heavy chain variable region comprisingcomplementarity determining regions CDR-H1, CDR-H2 and CDR-H3, the lightchain variable region comprising complementarity determining regionCDR-L1, CDR-L2 and CDR-L3 and with the amino acid sequences of said CDRscomprising the sequences in Tables 9B, 9C, 9G, 9H or 9I, optionallywherein the antibody or binding fragment is a monoclonal antibody, achimeric antibody, a humanized antibody or a polyclonal antibody or abinding fragment of any of the foregoing optionally wherein the bindingfragment is an antibody binding fragment selected from Fab, Fab′,F(ab′)2, scFv, dsFv, ds-scFv, dimers, nanobodies, minibodies, diabodies,and multimers thereof.
 20. An immunoconjugate comprising the antibody orbinding fragment of claim 19 and a detectable label or cytotoxic agent,optionally, wherein the detectable label comprises a positron emittingradionuclide, optionally for use in subject imaging such as PET imaging.21. A composition or kit comprising the antibody or binding fragment ofclaim 19, an immunoconjugate comprising said antibody, or a nucleic acidencoding said antibody.
 22. A nucleic acid molecule encoding theantibody of claim 19, optionally comprised in a vector, or a cellexpressing said antibody or binding fragment.
 23. A method, the methodcomprising contacting a biological sample, optionally a brain tissuesample or an extract thereof, whole blood, plasma, serum and/or CSF,from a subject with the antibody or binding fragment of claim 17 or animmunoconjugate comprising said antibody or binding fragment underconditions permissive for forming an antibody: A-beta oligomer complex;and detecting the presence of any antibody: A-beta oligomer complex. 24.The method of claim 23, wherein the level of A-beta is detected by SPR.